Cell expansion

ABSTRACT

Embodiments described herein generally provide for expanding cells in a cell expansion system. The cells may be grown in a bioreactor, and the cells may be activated by an activator (e.g., a soluble activator complex). Nutrient and gas exchange capabilities of a closed, automated cell expansion system may allow cells to be seeded at reduced cell seeding densities, for example. Parameters of the cell growth environment may be manipulated to load the cells into a particular position in the bioreactor for the efficient exchange of nutrients and gases. System parameters may be adjusted to shear any cell colonies that may form during the expansion phase. Metabolic concentrations may be controlled to improve cell growth and viability. Cell residence in the bioreactor may be controlled. In embodiments, the cells may include T cells. In further embodiments, the cells may include T cell subpopulations, including regulatory T cells (Tregs), for example.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to, and the benefit of, the followingapplications: U.S. Provisional Application Ser. No. 62/479,721, filed onMar. 31, 2017, and entitled, “Expanding Cells;” U.S. ProvisionalApplication Ser. No. 62/479,760, filed on Mar. 31, 2017, and entitled“Cell Expansion;” U.S. Provisional Application Ser. No. 62/479,788,filed on Mar. 31, 2017, and entitled, “Expanding Cells in a Bioreactor;”U.S. Provisional Application Ser. No. 62/549,871, filed on Aug. 24,2017, and entitled, “Expansion of Cells;” and U.S. ProvisionalApplication Ser. No. 62/647,361, filed on Mar. 23, 2018, and entitled,“Expansion of Cells.” The disclosures of the above-identifiedapplications are hereby incorporated by reference in their entireties asif set forth herein in full for all that they teach and for allpurposes.

BACKGROUND

Cell Expansion Systems (CESs) are used to expand and differentiatecells. Cell expansion systems may be used to expand, e.g., grow, avariety of adherent and suspension cells. Cells, of both adherent andnon-adherent type, may be grown in a bioreactor in a cell expansionsystem.

SUMMARY

Embodiments of the present disclosure generally relate to expandingcells in a cell expansion system (CES). Such expansion may occur throughthe use of a bioreactor or cell growth chamber, according toembodiments. In an embodiment, such bioreactor or cell growth chambermay comprise a hollow fiber membrane. Such hollow fiber membrane mayinclude an extracapillary (EC) space and an intracapillary (IC) space. Acell expansion system may expand a variety of cell types. Embodimentsmay provide for adherent or non-adherent cells to be grown or expandedin the cell expansion system. Non-adherent, or suspension, cells whichmay be expanded in the system may include T cells, or T lymphocytes, forexample. In embodiments, one or more subpopulations or subsets of Tcells may be grown. For example, embodiments may provide methods andsystems for expanding regulatory T cells (Tregs) or human regulatory Tcells (hTregs).

In embodiments, methods and systems may be provided for expanding cellsin a closed, automated cell expansion system. In an embodiment, suchcell expansion system may include a bioreactor or cell growth chamber.In further embodiments, such bioreactor or cell growth chamber maycomprise a hollow fiber membrane.

For example, non-adherent cells, e.g., T cells and/or Treg cells, may beintroduced into a hollow fiber bioreactor, in which the hollow fiberbioreactor comprises a plurality of hollow fibers. According to anembodiment, the cells may be exposed to a stimulator or activator tostimulate or activate the expansion of the cells in the hollow fiberbioreactor.

A closed, automated cell expansion system comprising a bioreactor orcell growth chamber may comprise numerous capabilities, such as nutrientand gas exchange capabilities. Such capabilities may allow cells to beseeded at reduced cell seeding densities as compared to cell seedingdensities used in culture flasks or other static culture methods.Embodiments provide for parameters of the cell growth environment to bemanipulated to load or introduce cells into a position in the bioreactorfor the efficient exchange or delivery of nutrients and gases to thegrowing cells. In embodiments, communication between the cells may alsobe promoted. For example, in an embodiment, a centralization, orcentering, of cells in the bioreactor may increase cell density topromote communication, such as chemical signaling, between cells.

Embodiments also may provide for system parameters to be managed tocontrol cell residence in the bioreactor or cell growth chamber. Bycontrolling cell residence in the bioreactor during the cell growthphase, for example, the system may provide for an efficient gas andnutrient exchange to expanding cells. In embodiments, a bioreactor maybe designed to provide gas exchange, and, in some embodiments, nutrientexchange, to growing cells. In an example embodiment, a bioreactorcomprising a semi-permeable hollow fiber membrane provides gas andnutrient exchange through the semi-permeable hollow fiber membrane. Thesemi-permeable hollow fibers of the bioreactor allow essential nutrients(e.g., glucose and/or cell growth formulated media) to reach the cellsand metabolic waste products (e.g., lactate) to exit the system viadiffusion through the walls of the hollow fibers. However, according toembodiments, cells residing in the headers of the bioreactor or in otherareas outside the hollow fibers may not receive proper gas exchange andnutrient exchange, which may result in cell aggregation and death.Embodiments therefore relate to providing methods to retain cellpopulations, e.g., non-adherent cell populations, in the hollow fibersof the bioreactor when feeding the cells. Such retaining of the cells inthe bioreactor may also promote cell-to-cell communications because withmore cells in the bioreactor itself, cell densities may increase andcell-to-cell communications may therefore improve.

Additional embodiments may provide for system features to be harnessedto shear any cell colonies, micro-colonies, or cell clusters that mayform during the expansion phase of cell growth. For example, anembodiment provides for, after a period of expansion, the shearing ofgroups of cells, e.g., colonies, micro-colonies, or clusters, through abioreactor hollow fiber membrane. Such shearing may reduce the number ofcells in the micro-colony, colony, or cluster, in which a micro-colony,colony, or cluster may be a group of one or more attached cells.Embodiments may provide a protocol to shear any colonies by circulatingthe suspension cell culture through, for example, the hollow fiberIntracapillary (IC) loop (e.g., with hollow fibers of 215 μm innerdiameter) during the expansion phase of growth. In embodiments, acolony, micro-colony, or cluster of cells may be sheared to reduce asize of the colony, micro-colony, or cluster of cells. In an embodiment,a colony, micro-colony, or cluster of cells may be sheared to provide asingle cell uniform suspension and improve cell growth/viability. Suchcapabilities may contribute to the continuous perfusion growth of thecells, e.g., T cells or Tregs.

Embodiments of the present disclosure further relate to growing cellsusing a combination of features for cell expansion. For example, in anembodiment, cells may be seeded at a reduced seeding density as comparedto a cell seeding density used in culture flasks or other static culturemethods. For example, in some embodiments, the volume of fluid used toseed the cells may include from about 1×10⁴ cells/mL to about 1×10⁶cell/mL, such as on the order of 10⁵ cells/mL. In other embodiments, thevolume of fluid used to seed the cells may include less than about 1×10⁶cells/mL. According to embodiments, the cells may be loaded, orintroduced, into a desired position in a bioreactor, e.g., hollow fiberbioreactor, in the cell expansion system. In an embodiment, cells may beexposed to an activator to activate expansion of the cells in the hollowfiber bioreactor, for example. Any colonies or clusters of cells thatmay form during cell expansion may be sheared by circulating the cellsto cause the cells to incur a shear stress and/or shear force, forexample, according to an embodiment. Such shear stress and/or shearforce may cause one or more cells to break apart from a cell colony, forexample. To provide proper gas and nutrient exchange to growing cellpopulations, further embodiments provide for retaining cell populations,e.g., suspension cell populations, inside the hollow fibers of thebioreactor during the feeding of the cells, for example. By centralizingand/or retaining cells in the hollow fibers of the bioreactor during thecell growth phase, cell densities may increase and cell-to-cellcommunications may improve. Reduced cell seeding densities, as comparedto static cultures, may therefore be used.

Embodiments further provide for a cell expansion system for expandingcells, in which such system may include, for example, a hollow fiberbioreactor comprising an inlet port and an outlet port; a first fluidflow path having at least opposing ends, in which a first opposing endof the first fluid flow path is fluidly associated with an inlet port ofthe bioreactor, and a second end of the first fluid flow path is fluidlyassociated with an outlet port of the bioreactor, wherein the firstfluid flow path is fluidly associated with an intracapillary portion ofthe bioreactor; a fluid inlet path fluidly associated with the firstfluid flow path; and a first fluid circulation path fluidly associatedwith the first fluid flow path and the intracapillary portion of thebioreactor.

Embodiments further provide for the cell expansion system to include aprocessor for executing instructions to perform methods described and/orillustrated herein. For example, embodiments of the present disclosureprovide for implementing such expansion of cells through the use of oneor more protocols or tasks for use with a cell expansion system. Forexample, such protocols or tasks may include pre-programmed protocols ortasks. In embodiments, a pre-programmed, default, or otherwisepreviously saved task may be selected by a user or system operator toperform a specific function by the cell expansion system. In otherembodiments, such protocols or tasks may include custom or user-definedprotocols or tasks. For example, through a user interface (UI) and oneor more graphical user interface (GUI) elements, a custom oruser-defined protocol or task may be created. A task may comprise one ormore steps.

As used herein, “at least one,” “one or more,” and “and/or” areopen-ended expressions that are both conjunctive and disjunctive inoperation. For example, each of the expressions “at least one of A, Band C,” “at least one of A, B, or C,” “one or more of A, B, and C,” “oneor more of A, B, or C” and “A, B, and/or C” means A alone, B alone, Calone, A and B together, A and C together, B and C together, or A, B andC together.

This Summary is included to provide a selection of concepts in asimplified form, in which such concepts are further described below inthe Detailed Description. This Summary is not intended to be used in anyway to limit the claimed subject matter's scope. Features, includingequivalents and variations thereof, may be included in addition to thoseprovided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present disclosure may be described by referencingthe accompanying figures. In the figures, like numerals refer to likeitems. In the figures, dashed lines may be used to indicate that anelement(s) may be optional.

FIG. 1A depicts an embodiment of a cell expansion system (CES).

FIG. 1B illustrates a front elevation view of an embodiment of abioreactor showing circulation paths through the bioreactor.

FIG. 1C depicts a rocking device for moving a cell growth chamberrotationally or laterally during operation of a cell expansion system,according to embodiments of the present disclosure.

FIG. 2 illustrates a perspective view of a cell expansion system with apremounted fluid conveyance device, in accordance with embodiments ofthe present disclosure.

FIG. 3 depicts a perspective view of a housing of a cell expansionsystem, in accordance with embodiments of the present disclosure.

FIG. 4 illustrates a perspective view of a premounted fluid conveyancedevice, in accordance with embodiments of the present disclosure.

FIG. 5A depicts a schematic of a cell expansion system, including anoperational configuration showing fluid movement, in accordance with anembodiment of the present disclosure.

FIG. 5B depicts a schematic of a cell expansion system, includinganother operational configuration showing fluid movement, in accordancewith an embodiment of the present disclosure.

FIG. 5C depicts a schematic of a cell expansion system, includinganother operational configuration showing fluid movement, in accordancewith an embodiment of the present disclosure.

FIG. 6 illustrates a schematic of a cell expansion system, in accordancewith another embodiment of the present disclosure.

FIG. 7 depicts a flow diagram illustrating the operationalcharacteristics of a process for expanding cells, in accordance withembodiments of the present disclosure.

FIG. 8A illustrates a flow diagram depicting the operationalcharacteristics of a process for expanding cells, in accordance withembodiments of the present disclosure.

FIG. 8B depicts a schematic of a portion of a cell expansion system, inaccordance with an embodiment of the present disclosure.

FIG. 9A depicts a flow diagram illustrating the operationalcharacteristics of a process for expanding cells, in accordance withembodiments of the present disclosure.

FIG. 9B illustrates a graph of oxygen consumption in a cell expansionsystem, in accordance with embodiments of the present disclosure.

FIG. 10A illustrates a flow diagram depicting the operationalcharacteristics of a process for expanding cells, in accordance withembodiments of the present disclosure.

FIG. 10B illustrates a table with example pump rates that may be used ina cell expansion system, in accordance with embodiments of the presentdisclosure.

FIG. 11A depicts a flow diagram illustrating the operationalcharacteristics of a process for expanding cells, in accordance withembodiments of the present disclosure.

FIG. 11B illustrates a graph of the metabolism of expanding cells, inaccordance with embodiments of the present disclosure.

FIG. 11C illustrates a graph of the metabolism of expanding cells, inaccordance with embodiments of the present disclosure.

FIG. 12 illustrates a flow diagram depicting the operationalcharacteristics of a process for expanding cells, in accordance withembodiments of the present disclosure.

FIG. 13 depicts a flow diagram illustrating the operationalcharacteristics of a process for expanding cells, in accordance withembodiments of the present disclosure.

FIG. 14 illustrates a flow diagram depicting the operationalcharacteristics of a process for expanding cells, in accordance withembodiments of the present disclosure.

FIG. 15A illustrates a flow diagram depicting the operationalcharacteristics of a process for expanding cells, in accordance withembodiments of the present disclosure.

FIG. 15B illustrates a graph of cell number versus flow rate during cellexpansion, in accordance with embodiments of the present disclosure.

FIG. 16 depicts a flow diagram illustrating the operationalcharacteristics of a process for expanding cells, in accordance withembodiments of the present disclosure.

FIG. 17A depicts a flow diagram illustrating the operationalcharacteristics of a process for expanding cells, in accordance withembodiments of the present disclosure.

FIG. 17B depicts views of cells expanding in a cell expansion system, inaccordance with embodiments of the present disclosure.

FIG. 17C illustrates a graph showing inner diameters of celldisassociation, in accordance with embodiments of the presentdisclosure.

FIG. 18 illustrates a graph of cell numbers and flow rate versus culturedays during cell expansion, in accordance with embodiments of thepresent disclosure.

FIG. 19A illustrates a flow diagram depicting the operationalcharacteristics of a process for operating pumps to expand cells, inaccordance with embodiments of the present disclosure.

FIG. 19B depicts a schematic of a portion of a cell expansion system, inaccordance with an embodiment of the present disclosure.

FIG. 19C depicts a schematic of a portion of a cell expansion system, inaccordance with an embodiment of the present disclosure.

FIG. 19D depicts a schematic of a portion of a cell expansion system, inaccordance with an embodiment of the present disclosure.

FIG. 20 illustrates a flow diagram depicting the operationalcharacteristics of a process for expanding cells, in accordance withembodiments of the present disclosure.

FIG. 21 depicts a flow diagram illustrating the operationalcharacteristics of a process for expanding cells, in accordance withembodiments of the present disclosure.

FIG. 22 illustrates a flow diagram depicting the operationalcharacteristics of a process for expanding cells, in accordance withembodiments of the present disclosure.

FIG. 23 depicts a flow diagram illustrating the operationalcharacteristics of a process for expanding cells, in accordance withembodiments of the present disclosure.

FIG. 24 depicts an example processing system of a cell expansion systemupon which embodiments of the present disclosure may be implemented.

DETAILED DESCRIPTION

The following Detailed Description provides a discussion of illustrativeembodiments with reference to the accompanying drawings. The inclusionof specific embodiments herein should not be construed as limiting orrestricting the present disclosure. Further, while language specific tofeatures, acts, and/or structures, for example, may be used indescribing embodiments herein, the claims are not limited to thefeatures, acts, and/or structures described. A person of skill in theart will appreciate that other embodiments, including improvements, arewithin the spirit and scope of the present disclosure. Further, anyalternatives or additions, including any listed as separate embodiments,may be used or incorporated with any other embodiments herein described.

Embodiments of the present disclosure are generally directed to systemsand methods for expanding cells in a cell expansion system (CES). Suchexpansion may occur through the use of a bioreactor or cell growthchamber, according to embodiments. In an embodiment, such bioreactor orcell growth chamber may comprise a hollow fiber membrane. Such hollowfiber membrane may include a plurality of hollow fibers and may includean extracapillary (EC) and/or intracapillary (IC) space. Embodiments mayprovide for adherent or non-adherent cells to be grown or expanded inthe cell expansion system. For example, non-adherent or suspensioncells, such as T cells, or T lymphocytes, may be expanded in the system.In embodiments, one or more subpopulations or subsets of T cells may begrown. For example, embodiments may provide methods and systems forexpanding regulatory T cells (Tregs) and/or human regulatory T cells(hTregs).

In embodiments, methods and systems may be provided for expanding cellsin a closed, automated cell expansion system. In an embodiment, suchcell expansion system may include a bioreactor or cell growth chamber.In further embodiments, such bioreactor or cell growth chamber maycomprise a hollow fiber membrane. The capabilities of such system, suchas nutrient and gas exchange capabilities, may allow cells to be seededat reduced cell seeding densities. Embodiments provide for parameters ofthe cell growth environment to be manipulated to load or introduce cellsinto a position in the bioreactor for the efficient exchange ofnutrients and gases to the growing cells. For example, in an embodiment,the centralization of cells in the bioreactor may increase cell density.

In embodiments, non-adherent cell populations, e.g., T cells and/or Tregcells, may be introduced or loaded into a hollow fiber bioreactor, inwhich the hollow fiber bioreactor may comprise a plurality of hollowfibers. In embodiments, the cells may be exposed to an activator toactivate the expansion of the cells in the hollow fiber bioreactor. Inan embodiment, a plurality of cells may be introduced into a cellexpansion system using a “load cells centrally without circulation”task, for example. Such task may be performed on Day 0 and on Days 4-8,according to an example embodiment. Other days may be used in otherembodiments. In embodiments, such loading of cells task may result inthe centralization of cells in the bioreactor to increase cell density.In other embodiments, the cells may be located in other portions orregions of the bioreactor to increase cell density. In embodiments, bypositioning the cells in a first position, e.g., about a central region,of the bioreactor, the cells may receive an efficient exchange ofnutrients and gases.

In embodiments, a reduced cell seeding density, as compared to cellseeding densities used with static culture methods, may be used. In anembodiment using a cell expansion system, cells, e.g., Tregs or Tregcells, may be expanded from a cell seeding density of 2.54×10⁵ cells/mLto 3.69×10⁵ cells/mL. In other embodiments, the cell seeding density maybe less than about 1×10⁶ cells/mL. In addition, a Treg cell inoculum maybe prepared from a cell seeding density of 1.0×10⁵ cells/mL. Othermethods, e.g., static Treg cell culture methods, may use a cell seedingdensity of 1.0×10⁶ Treg cells/mL for in vitro expansion. In anembodiment, lower cell seeding densities may be used due to the system'soverall efficiency in delivering nutrients to the culture environment,for example. In other embodiments, one or more of the steps used duringexpansion in combination with the system's overall efficiency indelivering nutrients to the culture environment may allow lower initialcell seeding densities to be used.

In embodiments, an automated cell, e.g., Treg, expansion may beperformed with a soluble activator complex. In other embodiments, othertypes of activators, such as beads for the stimulation of cells, may beused, for example. In other embodiments, cells, e.g., Treg cells, may beexpanded without the use of a bead-based stimulation. In one embodiment,cell expansion may be performed using the Stem Cell Technologies solubleImmunoCult™ Human CD3/CD28/CD2 T Cell Activator to activate and expandTreg cells in the presence of 200 IU/mL of the cytokine IL-2 with anautomated cell expansion system. Using a soluble activator complex mayreduce costs for stimulation over the cost of a bead-based protocol, forexample. Other types of activators may be used in other embodiments.Further, other types of cytokines or other growth factors may be used inother embodiments.

Embodiments also may provide for system parameters to be adjusted ormanaged to control cell residence in the bioreactor or cell growthchamber. By controlling cell residence in the bioreactor hollow fibersduring the cell growth phase, for example, the system may provide for anefficient gas and nutrient exchange to expanding cells. In embodiments,a bioreactor may be designed to provide gas exchange, and, in someembodiments, nutrient exchange, to growing cells. In an exampleembodiment, a bioreactor comprising a semi-permeable hollow fibermembrane may provide gas and nutrient exchange through thesemi-permeable hollow fiber membrane. In embodiments, methods forproviding media constituents, such as various cytokines, proteins, etc.,for example, to growing cells which cannot pass through the membrane mayuse a fluid inlet to the side of the bioreactor, e.g., intracapillary(IC) side, where cells are growing. However, even low, decreased, orreduced, e.g., minimum, inlet flow rates (0.1 mL/min, for example) mayresult in cells collecting in the outlet header of the bioreactor,according to embodiments. Cells residing in the headers of thebioreactor may not receive proper gas exchange and nutrient exchange,which may result in cell death and aggregation.

Embodiments relate to providing methods to retain cells, e.g.,non-adherent cell populations, in the bioreactor when feeding the cellsusing an inlet, e.g., IC inlet, flow. While embodiments herein may referto cells being on the IC side of the membrane when feeding, for example,other embodiments may provide for the cells to be on the EC side of themembrane, in which cells may be contained within a first circulationpath and/or a second circulation path, according to embodiments. Inembodiments, a feeding method may provide for pumping a first volume offluid, e.g., media or cell growth formulated media, into a first port ofthe bioreactor at a first volumetric flow rate, volume flow rate, fluidflow rate, flow rate, rate of fluid flow, or volume velocity, forexample. Volumetric flow rate, volume flow rate, fluid flow rate, flowrate, rate of fluid flow, or volume velocity, for example, may be usedinterchangeably. In some embodiments, flow rate may be a vector havingboth a speed and a direction. A second volume of the fluid may be pumpedinto a second port of the bioreactor at a second volumetric flow rate,volume flow rate, fluid flow rate, flow rate, rate of fluid flow, orvolume velocity. In embodiments, such volumetric flow rate, volume flowrate, fluid flow rate, flow rate, rate of fluid flow, or volumevelocity, may be controlled by one or more pump rate(s) and/or pump flowrate(s), for example. A pump rate may produce, cause, or affect avolumetric flow rate or flow rate of a fluid upon which the pump mayact. As used herein, a pump rate may be described in embodiments as thevolumetric flow rate or fluid flow rate produced, caused, or affected bythe pump.

In embodiments, the second flow rate of the fluid into the bioreactormay be opposite the direction of the first flow rate of the fluid intothe bioreactor. For example, FIGS. 5B and 5C illustrate exampleoperational configurations showing flow rates and flow directions thatmay be used with a cell expansion system, such as CES 500 (e.g., FIGS.5B, & 5C), in accordance with embodiments of the present disclosure. Inembodiments, cell expansion system pumps, e.g., IC pumps, may be used tocontrol cell residence in the bioreactor. In embodiments, cells may belost from the bioreactor into the IC circulation path or IC loop, forexample, during the expansion phase of growth. In embodiments, cells inthe bioreactor that are closer to IC inlet port, for example, mayreceive the freshest growth media, whereas, cells in the portion of theIC circulation path outside of the bioreactor, for example, mayessentially be receiving expended or conditioned media which may affecttheir glycolytic metabolism. In addition, cells in the bioreactor mayreceive mixed gas (O₂, CO₂, N₂) input from the gas transfer module (GTM)by diffusion from the EC loop circulation, whereas cells in the portionof the IC circulation path outside of the bioreactor may not, accordingto embodiments.

In embodiments, reducing the loss of cells from a hollow fiber membrane(HFM) bioreactor may be accomplished by matching, or closely orsubstantially matching, the IC circulation pump rate to the IC inletpump rate, but in the opposite direction, during feeding. For example,an IC inlet pump rate of +0.1 mL/min may be matched, or closely orsubstantially matched, to a complementary IC circulation pump rate of−0.1 mL/min in order to maintain cells in the bioreactor during thegrowth phase of the cell culture which may be Days 4-7, in embodiments.This pump adjustment may counteract the forces associated with the lossof cells from the IC outlet port, in accordance with embodiments. Inother embodiments, other pump rates may be used. For example, in otherembodiments, the pump rates may be different. In embodiments, otherpumps or additional pumps may be used. In an embodiment, fewer pumps maybe used. Further, other time periods may be used in other embodiments.

In embodiments, the metabolic activity of the cell population may affectfeeding parameters. For example, cell culture lactate values may bemaintained at or below a predefined level. In an embodiment, cellculture values may be maintained at or below about 7 mmol/L, forexample. In embodiments, by using a cell expansion system graphical userinterface (GUI) to control a rate(s) of media addition, lactatemetabolic waste product from glycolysis may be maintained at or below apredefined value, during the expansion of cells, e.g., regulatory Tcells. In other embodiments, rate(s) of media addition, for example,and/or other settings may be controlled to maintain, or attempt tomaintain, the lactate levels≤about 5 mmol/L, for example, to improvecell growth and viability. Other concentrations may be used in otherembodiments.

Additional embodiments may provide for system features to be harnessedto shear any cell colonies, micro-colonies, or cell clusters that mayform during the expansion phase. For example, an embodiment provides forthe shearing of colonies, e.g., micro-colonies, of cells through abioreactor hollow fiber membrane to reduce the number of cells in themicro-colony, colony, or cluster, in which a micro-colony, colony, orcluster may be a group of one or more attached cells. In embodiments, acell expansion system (CES) bioreactor architecture may be used to shearcell, e.g., Treg cell, micro-colonies. In embodiments, as cells, e.g.,Treg cells, grow, they tend to form micro-colonies that may limit thediffusion of nutrients to the cell(s) in the center of the colony. Thismay lead to adverse effects such as necrosis during cell culture.Embodiments may provide a protocol to shear the colonies by circulatingthe suspension cell culture through, for example, the hollow fiberIntracapillary (IC) loop (e.g., with hollow fibers of 215 μm innerdiameter) during the expansion phase of growth. In embodiments, acolony, micro-colony, or cluster of cells may be sheared to reduce asize of the colony, micro-colony, or cluster of cells. In an embodiment,a colony or cluster of cells may be sheared to provide a single cellsuspension and create cell growth/viability. In embodiments, suchcapabilities may contribute to the continuous perfusion growth of thecells, e.g., T cells or Tregs.

In embodiments, a therapeutic dose of cells, e.g., Tregs, may beexpanded in, and harvested from, a cell expansion system. Inembodiments, the number of cells at harvest may be from about 1×10⁶cells to about 1×10¹⁰ cells, such as on the order of 1×10⁹ cells. In oneembodiment, the number of harvested cells may be from about 1×10⁸ and1×10¹⁰ cells, one example being between about 7.0×10⁸ to about 1.4×10⁹cells. In embodiments, the harvested cells may have viabilities betweenabout 60% and about 100%. For example, the viability of the harvestedcells may be above about 65%, above about 70%, above about 75%, aboveabout 80%, above about 85%, above about 90%, or even above about 95%.The harvested cells may express biomarkers consistent with Tregs, insome embodiments. For example, the cells may express CD4⁺, CD25⁺, and/orFoxP3⁺ biomarkers, in some embodiments. In embodiments, the harvestedcells may include the CD4+CD25+ phenotype at a frequency of betweenabout 50% and about 100%. The harvested cells may include the CD4⁺CD25⁺phenotype at a frequency of above about 75%, above about 80%, aboveabout 85%, above about 90%, or even above about 95%. In otherembodiments, the cells may include the CD4⁺FoxP3⁺ phenotype at afrequency of between about 30% to about 100%. In some embodiments, theharvested cells may include the CD4⁺FoxP3⁺ phenotype at a frequency ofabove about 30%, above about 35%, above about 40%, above about 45%,above about 50%, above about 55%, above about 60%, above about 65%, oreven above about 70%.

Embodiments are directed to a cell expansion system, as noted above. Inembodiments, such cell expansion system is closed, in which a closedcell expansion system comprises contents that are not directly exposedto the atmosphere. Such cell expansion system may be automated. Inembodiments, cells, of both adherent and non-adherent or suspensiontype, may be grown in a bioreactor in the cell expansion system.According to embodiments, the cell expansion system may include basemedia or other type of media. Methods for replenishment of media areprovided for cell growth occurring in a bioreactor of the closed cellexpansion system. In embodiments, the bioreactor used with such systemsis a hollow fiber bioreactor. Many types of bioreactors may be used inaccordance with embodiments of the present disclosure.

The system may include, in embodiments, a bioreactor that is fluidlyassociated with a first fluid flow path having at least opposing ends, afirst opposing end of the first fluid flow path fluidly associated witha first port of a hollow fiber membrane and a second end of the firstfluid flow path fluidly associated with a second port of the hollowfiber membrane. In embodiments, a hollow fiber membrane comprises aplurality of hollow fibers. The system may further include a fluid inletpath fluidly associated with the first fluid flow path, in which aplurality of cells may be introduced into the first fluid flow paththrough the first fluid inlet path. In some embodiments, a pump fortransferring intracapillary inlet fluid from an intracapillary media bagto the first fluid flow path and a controller for controlling operationof the pump are included. The controller, in embodiments, controls thepump to transfer cells from a cell inlet bag to the first fluid flowpath, for example. Another pump for circulating fluid in a first fluidcirculation path may also be included, in which such pump may alsoinclude a controller for controlling operation of the pump. In anembodiment, a controller is a computing system, including aprocessor(s), for example. The one or more controller(s) may beconfigured, in embodiments, to control the one or more pump(s), such asto circulate a fluid at a flow rate within the first fluid circulationpath, for example. A number of controllers may be used, e.g., a firstcontroller, second controller, third controller, fourth controller,fifth controller, sixth controller, etc., in accordance withembodiments. Further, a number of pumps may be used, e.g., a first pump,second pump, third pump, fourth pump, fifth pump, sixth pump, etc., inaccordance with embodiments of the present disclosure. In addition,while the present disclosure may refer to a media bag, a cell inlet bag,etc., multiple bags, e.g., a first media bag, a second media bag, athird media bag, a first cell inlet bag, a second cell inlet bag, athird cell inlet bag, etc., and/or other types of containers, may beused in embodiments. In other embodiments, a single media bag, a singlecell inlet bag, etc., may be used. Further, additional or other fluidpaths, e.g., a second fluid flow path, a second fluid inlet path, asecond fluid circulation path, etc., may be included in embodiments.

In embodiments, the system is controlled by, for example: a processorcoupled to the cell expansion system; a display device, in communicationwith the processor, and operable to display data; and a memory, incommunication with and readable by the processor, and containing aseries of instructions. In embodiments, when the instructions areexecuted by the processor, the processor receives an instruction toprime the system, for example. In response to the instruction to primethe system, the processor may execute a series of steps to prime thesystem and may next receive an instruction to perform an IC/EC washout,for example. In response to an instruction to load cells, for example,the processor may execute a series of steps to load the cells from acell inlet bag, for example, into the bioreactor.

A schematic of an example cell expansion system (CES) is depicted inFIG. 1A, in accordance with embodiments of the present disclosure. CES10 includes first fluid circulation path 12 and second fluid circulationpath 14. First fluid flow path 16 has at least opposing ends 18 and 20fluidly associated with a hollow fiber cell growth chamber 24 (alsoreferred to herein as a “bioreactor”), according to embodiments.Specifically, opposing end 18 may be fluidly associated with a firstinlet 22 of cell growth chamber 24, and opposing end 20 may be fluidlyassociated with first outlet 28 of cell growth chamber 24. Fluid infirst circulation path 12 flows through the interior of hollow fibers116 (see FIG. 1B) of hollow fiber membrane 117 (see FIG. 1B) disposed incell growth chamber 24 (cell growth chambers and hollow fiber membranesare described in more detail infra). Further, first fluid flow controldevice 30 may be operably connected to first fluid flow path 16 and maycontrol the flow of fluid in first circulation path 12.

Second fluid circulation path 14 includes second fluid flow path 34,cell growth chamber 24, and a second fluid flow control device 32. Thesecond fluid flow path 34 has at least opposing ends 36 and 38,according to embodiments. Opposing ends 36 and 38 of second fluid flowpath 34 may be fluidly associated with inlet port 40 and outlet port 42respectively of cell growth chamber 24. Fluid flowing through cellgrowth chamber 24 may be in contact with the outside of hollow fibermembrane 117 (see FIG. 1B) in the cell growth chamber 24, in which ahollow fiber membrane comprises a plurality of hollow fibers. Secondfluid circulation path 14 may be operably connected to second fluid flowcontrol device 32.

First and second fluid circulation paths 12 and 14 may thus be separatedin cell growth chamber 24 by a hollow fiber membrane 117 (see FIG. 1B).Fluid in first fluid circulation path 12 flows through theintracapillary (“IC”) space of the hollow fibers in the cell growthchamber 24. First circulation path 12 may be referred to as the “ICloop.” Fluid in second circulation path 14 flows through theextracapillary (“EC”) space in the cell growth chamber 24. Second fluidcirculation path 14 may be referred to as the “EC loop.” Fluid in firstfluid circulation path 12 may flow in either a co-current orcounter-current direction with respect to flow of fluid in second fluidcirculation path 14, according to embodiments.

Fluid inlet path 44 may be fluidly associated with first fluidcirculation path 12. Fluid inlet path 44 allows fluid into first fluidcirculation path 12, while fluid outlet path 46 allows fluid to leaveCES 10. Third fluid flow control device 48 may be operably associatedwith fluid inlet path 44. Alternatively, third fluid flow control device48 may alternatively be associated with first outlet path 46.

Fluid flow control devices as used herein may comprise a pump, valve,clamp, or combination thereof, according to embodiments. Multiple pumps,valves, and clamps can be arranged in any combination. In variousembodiments, the fluid flow control device is or includes a peristalticpump. In embodiments, fluid circulation paths, inlet ports, and outletports may be constructed of tubing of any material.

Various components are referred to herein as “operably associated.” Asused herein, “operably associated” refers to components that are linkedtogether in operable fashion and encompasses embodiments in whichcomponents are linked directly, as well as embodiments in whichadditional components are placed between the two linked components.“Operably associated” components can be “fluidly associated.” “Fluidlyassociated” refers to components that are linked together such thatfluid can be transported between them. “Fluidly associated” encompassesembodiments in which additional components are disposed between the twofluidly associated components, as well as components that are directlyconnected. Fluidly associated components can include components that donot contact fluid, but contact other components to manipulate the system(e.g., a peristaltic pump that pumps fluids through flexible tubing bycompressing the exterior of the tube).

Generally, any kind of fluid, including buffers, protein containingfluid, and cell-containing fluid, for example, can flow through thevarious circulations paths, inlet paths, and outlet paths. As usedherein, “fluid,” “media,” and “fluid media” are used interchangeably.

Turning to FIG. 1B, an example of a hollow fiber cell growth chamber 100which may be used with the present disclosure is shown in front sideelevation view. Cell growth chamber 100 has a longitudinal axis LA-LAand includes cell growth chamber housing 104. In at least oneembodiment, cell growth chamber housing 104 includes four openings orports: IC inlet port 108, IC outlet port 120, EC inlet port 128, and ECoutlet port 132.

According to embodiments of the present disclosure, fluid in a firstcirculation path enters cell growth chamber 100 through IC inlet port108 at a first longitudinal end 112 of the cell growth chamber 100,passes into and through the intracapillary side (referred to in variousembodiments as the intracapillary (“IC”) side or “IC space” of a hollowfiber membrane) of a plurality of hollow fibers 116 comprising hollowfiber membrane 117, and out of cell growth chamber 100 through IC outletport 120 located at a second longitudinal end 124 of the cell growthchamber 100. The fluid path between the IC inlet port 108 and the ICoutlet port 120 defines the IC portion 126 of the cell growth chamber100. Fluid in a second circulation path flows in the cell growth chamber100 through EC inlet port 128, comes in contact with the extracapillaryside or outside (referred to as the “EC side” or “EC space” of themembrane) of the hollow fibers 116, and exits cell growth chamber 100via EC outlet port 132. The fluid path between the EC inlet port 128 andthe EC outlet port 132 comprises the EC portion 136 of the cell growthchamber 100. Fluid entering cell growth chamber 100 via the EC inletport 128 may be in contact with the outside of the hollow fibers 116.Small molecules (e.g., ions, water, oxygen, lactate, etc.) may diffusethrough the hollow fibers 116 from the interior or IC space of thehollow fiber to the exterior or EC space, or from the EC space to the ICspace. Large molecular weight molecules, such as growth factors, may betypically too large to pass through the hollow fiber membrane, and mayremain in the IC space of the hollow fibers 116. The media may bereplaced as needed, in embodiments. Media may also be circulated throughan oxygenator or gas transfer module to exchange gasses as needed. Cellsmay be contained within a first circulation path and/or a secondcirculation path, as described below, and may be on either the IC sideand/or EC side of the membrane, according to embodiments.

The material used to make the hollow fiber membrane 117 may be anybiocompatible polymeric material which is capable of being made intohollow fibers. One material which may be used is a syntheticpolysulfone-based material, according to an embodiment of the presentdisclosure.

In embodiments, the CES (such as CES 500 (see FIGS. 5A, 5B, & 5C) and/orCES 600 (see FIG. 6), for example) may include a device configured tomove or “rock” the cell growth chamber relative to other components ofthe cell expansion system by attaching it to a rotational and/or lateralrocking device. FIG. 1C shows one such device, in which a bioreactor 100may be rotationally connected to two rotational rocking components andto a lateral rocking component, according to an embodiment.

A first rotational rocking component 138 rotates the bioreactor 100around central axis 142 of the bioreactor 100. Rotational rockingcomponent 138 may be rotationally associated with bioreactor 100. Inembodiments, bioreactor 100 may be rotated continuously in a singledirection around central axis 142 in a clockwise or counterclockwisedirection. Alternatively, bioreactor 100 may rotate in alternatingfashion, first clockwise, then counterclockwise, for example, aroundcentral axis 142, according to embodiments.

The CES may also include a second rotational rocking component thatrotates bioreactor 100 around rotational axis 144. Rotational axis 144may pass through the center point of bioreactor 100 and may be normal tocentral axis 142. Bioreactor 100 may be rotated continuously in a singledirection around rotational axis 144 in a clockwise or counterclockwisedirection, in embodiments. Alternatively, bioreactor 100 may be rotatedaround rotational axis 144 in an alternating fashion, first clockwise,then counterclockwise, for example. In various embodiments, bioreactor100 may also be rotated around rotational axis 144 and positioned in ahorizontal or vertical orientation relative to gravity.

In embodiments, lateral rocking component 140 may be laterallyassociated with bioreactor 100. The plane of lateral rocking component140 moves laterally in the −x and −y directions, in embodiments. Thesettling of cells in the bioreactor may be reduced by movement ofcell-containing media within the hollow fibers, according toembodiments.

The rotational and/or lateral movement of a rocking device may reducethe settling of cells within the device and reduce the likelihood ofcells becoming trapped within a portion of the bioreactor. The rate ofcells settling in the cell growth chamber is proportional to the densitydifference between the cells and the suspension media, according toStokes's Law. In certain embodiments, a 180-degree rotation (fast) witha pause (having a total combined time of 30 seconds, for example)repeated as described above keeps non-adherent red blood cells, forexample, suspended. A minimum rotation of about 180 degrees would bepreferred in an embodiment; however, one could use rotation of up to 360degrees or greater. Different rocking components may be used separately,or may be combined in any combination. For example, a rocking componentthat rotates bioreactor 100 around central axis 142 may be combined withthe rocking component that rotates bioreactor 100 around axis 144.Likewise, clockwise and counterclockwise rotation around different axesmay be performed independently in any combination.

Turning to FIG. 2, an embodiment of a cell expansion system 200 with apremounted fluid conveyance assembly is shown in accordance withembodiments of the present disclosure. The CES 200 includes a cellexpansion machine 202 that comprises a hatch or closable door 204 forengagement with a back portion 206 of the cell expansion machine 202. Aninterior space 208 within the cell expansion machine 202 includesfeatures adapted for receiving and engaging a premounted fluidconveyance assembly 210. The premounted fluid conveyance assembly 210 isdetachably-attachable to the cell expansion machine 202 to facilitaterelatively quick exchange of a new or unused premounted fluid conveyanceassembly 210 at a cell expansion machine 202 for a used premounted fluidconveyance assembly 210 at the same cell expansion machine 202. A singlecell expansion machine 202 may be operated to grow or expand a first setof cells using a first premounted fluid conveyance assembly 210 and,thereafter, may be used to grow or expand a second set of cells using asecond premounted fluid conveyance assembly 210 without needing to besanitized between interchanging the first premounted fluid conveyanceassembly 210 for the second premounted fluid conveyance assembly 210.The premounted fluid conveyance assembly 210 includes a bioreactor 100and an oxygenator or gas transfer module 212 (also see FIG. 4). Tubingguide slots are shown as 214 for receiving various media tubingconnected to premounted fluid conveyance assembly 210, according toembodiments.

Next, FIG. 3 illustrates the back portion 206 of cell expansion machine202 prior to detachably-attaching a premounted fluid conveyance assembly210 (FIG. 2), in accordance with embodiments of the present disclosure.The closable door 204 (shown in FIG. 2) is omitted from FIG. 3. The backportion 206 of the cell expansion machine 202 includes a number ofdifferent structures for working in combination with elements of apremounted fluid conveyance assembly 210. More particularly, the backportion 206 of the cell expansion machine 202 includes a plurality ofperistaltic pumps for cooperating with pump loops on the premountedfluid conveyance assembly 210, including the IC circulation pump 218,the EC circulation pump 220, the IC inlet pump 222, and the EC inletpump 224. In addition, the back portion 206 of the cell expansionmachine 202 includes a plurality of valves, including the IC circulationvalve 226, the reagent valve 228, the IC media valve 230, the airremoval valve 232, the cell inlet valve 234, the wash valve 236, thedistribution valve 238, the EC media valve 240, the IC waste or outletvalve 242, the EC waste valve 244, and the harvest valve 246. Severalsensors are also associated with the back portion 206 of the cellexpansion machine 202, including the IC outlet pressure sensor 248, thecombination IC inlet pressure and temperature sensors 250, thecombination EC inlet pressure and temperature sensors 252, and the ECoutlet pressure sensor 254. Also shown is an optical sensor 256 for anair removal chamber, according to an embodiment.

In accordance with embodiments, a shaft or rocker control 258 forrotating the bioreactor 100 is shown. Shaft fitting 260 associated withthe shaft or rocker control 258 allows for proper alignment of a shaftaccess aperture, see e.g., 424 (FIG. 4) of a tubing-organizer, see e.g.,300 (FIG. 4) of a premounted conveyance assembly 210 or 400 with theback portion 206 of the cell expansion machine 202. Rotation of shaft orrocker control 258 imparts rotational movement to shaft fitting 260 andbioreactor 100. Thus, when an operator or user of the CES 200 attaches anew or unused premounted fluid conveyance assembly 400 (FIG. 4) to thecell expansion machine 202, the alignment is a relatively simple matterof properly orienting the shaft access aperture 424 (FIG. 4) of thepremounted fluid conveyance assembly 210 or 400 with the shaft fitting260.

Turning to FIG. 4, a perspective view of a detachably-attachablepremounted fluid conveyance assembly 400 is shown. The premounted fluidconveyance assembly 400 may be detachably-attachable to the cellexpansion machine 202 (FIGS. 2 and 3) to facilitate relatively quickexchange of a new or unused premounted fluid conveyance assembly 400 ata cell expansion machine 202 for a used premounted fluid conveyanceassembly 400 at the same cell expansion machine 202. As shown in FIG. 4,the bioreactor 100 may be attached to a bioreactor coupling thatincludes a shaft fitting 402. The shaft fitting 402 includes one or moreshaft fastening mechanisms, such as a biased arm or spring member 404for engaging a shaft, e.g., 258 (shown in FIG. 3), of the cell expansionmachine 202.

According to embodiments, the premounted fluid conveyance assembly 400includes tubing 408A, 408B, 408C, 408D, 408E, etc., and various tubingfittings to provide the fluid paths shown in FIGS. 5A, 5B, 5C, and 6, asdiscussed below. Pump loops 406A, 406B, and 406C may also be providedfor the pump(s). In embodiments, although the various media may beprovided at the site where the cell expansion machine 202 is located,the premounted fluid conveyance assembly 400 may include sufficienttubing length to extend to the exterior of the cell expansion machine202 and to enable welded connections to tubing associated with mediabag(s) or container(s), according to embodiments.

Next, FIGS. 5A, 5B, and 5C illustrate schematics of embodiments of acell expansion system 500, and FIG. 6 illustrates a schematic of anotherembodiment of a cell expansion system 600. In the embodiments shown inFIGS. 5A, 5B, 5C, and 6, and as described below, the cells are grown inthe IC space. However, the disclosure is not limited to such examplesand may in other embodiments provide for cells to be grown in the ECspace.

As noted, FIGS. 5A, 5B, and 5C illustrate a CES 500. While FIGS. 5A, 5B,and 5C depict substantially similar structural components of CES 500,FIGS. 5A, 5B, and 5C illustrate possible operational configurations offluid movement in a first fluid circulation path using the structuralfeatures of CES 500, in accordance with embodiments of the presentdisclosure. As shown, CES 500 includes first fluid circulation path 502(also referred to as the “intracapillary loop” or “IC loop”) and secondfluid circulation path 504 (also referred to as the “extracapillaryloop” or “EC loop”), according to embodiments. First fluid flow path 506may be fluidly associated with cell growth chamber 501 to form firstfluid circulation path 502. Fluid flows into cell growth chamber 501through IC inlet port 501A, through hollow fibers in cell growth chamber501, and exits via IC outlet port 501B. Pressure gauge 510 measures thepressure of media leaving cell growth chamber or bioreactor 501. Mediaflows through IC circulation pump 512 which may be used to control therate of media flow. IC circulation pump 512 may pump the fluid in afirst direction or second direction opposite the first direction. Exitport 501B may be used as an inlet in the reverse direction. For example,in a first configuration, the IC circulation pump may pump the fluid ina positive direction, in which the fluid enters the IC inlet port 501A.In a second configuration, for example, the IC circulation pump may pumpthe fluid in a negative direction, in which the fluid enters the ICoutlet port 501B, for example.

Media entering the IC loop may enter through valve 514. As those skilledin the art will appreciate, additional valves, pressure gauges,pressure/temperature sensors, ports, and/or other devices may be placedat various locations to isolate and/or measure characteristics of themedia along portions of the fluid paths. Accordingly, it is to beunderstood that the schematic shown represents one possibleconfiguration for various elements of the CES 500, and modifications tothe schematic shown are within the scope of the one or more presentembodiments.

With regard to the IC loop 502, samples of media may be obtained fromsample port 516 or sample coil 518 during operation.Pressure/temperature gauge 520 disposed in first fluid circulation path502 allows detection of media pressure and temperature during operation.Media then returns to IC inlet port 501A to complete fluid circulationpath 502. Cells grown/expanded in cell growth chamber 501 may be flushedout of cell growth chamber 501 into harvest bag 599 through valve 598 orredistributed within the hollow fibers for further growth.

Fluid in second fluid circulation path 504 enters cell growth chamber501 via EC inlet port 501C, and leaves cell growth chamber 501 via ECoutlet port 501D. Media in the EC loop 504 may be in contact with theoutside of the hollow fibers in the cell growth chamber 501, therebyallowing diffusion of small molecules into and out of the hollow fibers.

Pressure/temperature gauge 524 disposed in the second fluid circulationpath 504 allows the pressure and temperature of media to be measuredbefore the media enters the EC space of the cell growth chamber 501,according to an embodiment. Pressure gauge 526 allows the pressure ofmedia in the second fluid circulation path 504 to be measured after itleaves the cell growth chamber 501. With regard to the EC loop, samplesof media may be obtained from sample port 530 or a sample coil duringoperation.

In embodiments, after leaving EC outlet port 501D of cell growth chamber501, fluid in second fluid circulation path 504 passes through ECcirculation pump 528 to oxygenator or gas transfer module 532. ECcirculation pump 528 may also pump the fluid in opposing directions.Second fluid flow path 522 may be fluidly associated with oxygenator orgas transfer module 532 via oxygenator inlet port 534 and oxygenatoroutlet port 536. In operation, fluid media flows into oxygenator or gastransfer module 532 via oxygenator inlet port 534, and exits oxygenatoror gas transfer module 532 via oxygenator outlet port 536. Oxygenator orgas transfer module 532 adds oxygen to, and removes bubbles from, mediain the CES 500, for example. In various embodiments, media in secondfluid circulation path 504 may be in equilibrium with gas enteringoxygenator or gas transfer module 532. The oxygenator or gas transfermodule 532 may be any appropriately sized oxygenator or gas transferdevice. Air or gas flows into oxygenator or gas transfer module 532 viafilter 538 and out of oxygenator or gas transfer device 532 throughfilter 540. Filters 538 and 540 reduce or prevent contamination ofoxygenator or gas transfer module 532 and associated media. Air or gaspurged from the CES 500 during portions of a priming sequence may ventto the atmosphere via the oxygenator or gas transfer module 532.

In accordance with at least one embodiment, media, including cells (frombag 562), and fluid media from bag 546 may be introduced to first fluidcirculation path 502 via first fluid flow path 506. Fluid container 562(e.g., Cell Inlet Bag or Saline Priming Fluid for priming air out of thesystem) may be fluidly associated with the first fluid flow path 506 andthe first fluid circulation path 502 via valve 564.

Fluid containers, or media bags, 544 (e.g., Reagent) and 546 (e.g., ICMedia) may be fluidly associated with either first fluid inlet path 542via valves 548 and 550, respectively, or second fluid inlet path 574 viavalves 570 and 576. First and second sterile sealable input primingpaths 508 and 509 are also provided. An air removal chamber (ARC) 556may be fluidly associated with first circulation path 502. The airremoval chamber 556 may include one or more ultrasonic sensors includingan upper sensor and lower sensor to detect air, a lack of fluid, and/ora gas/fluid interface, e.g., an air/fluid interface, at certainmeasuring positions within the air removal chamber 556. For example,ultrasonic sensors may be used near the bottom and/or near the top ofthe air removal chamber 556 to detect air, fluid, and/or an air/fluidinterface at these locations. Embodiments provide for the use ofnumerous other types of sensors without departing from the spirit andscope of the present disclosure. For example, optical sensors may beused in accordance with embodiments of the present disclosure. Air orgas purged from the CES 500 during portions of the priming sequence orother protocols may vent to the atmosphere out air valve 560 via line558 that may be fluidly associated with air removal chamber 556.

EC media (e.g., from bag 568) or wash solution (e.g., from bag 566) maybe added to either the first or second fluid flow paths. Fluid container566 may be fluidly associated with valve 570 that may be fluidlyassociated with first fluid circulation path 502 via distribution valve572 and first fluid inlet path 542. Alternatively, fluid container 566may be fluidly associated with second fluid circulation path 504 viasecond fluid inlet path 574 and EC inlet path 584 by opening valve 570and closing distribution valve 572. Likewise, fluid container 568 may befluidly associated with valve 576 that may be fluidly associated withfirst fluid circulation path 502 via first fluid inlet path 542 anddistribution valve 572. Alternatively, fluid container 568 may befluidly associated with second fluid inlet path 574 by opening valve 576and closing distribution valve 572.

An optional heat exchanger 552 may be provided for media reagent or washsolution introduction.

In the IC loop, fluid may be initially advanced by the IC inlet pump554. In the EC loop, fluid may be initially advanced by the EC inletpump 578. An air detector 580, such as an ultrasonic sensor, may also beassociated with the EC inlet path 584.

In at least one embodiment, first and second fluid circulation paths 502and 504 are connected to waste line 588. When valve 590 is opened, ICmedia may flow through waste line 588 and to waste or outlet bag 586.Likewise, when valve 582 is opened, EC media may flow through waste line588 to waste or outlet bag 586.

In embodiments, cells may be harvested via cell harvest path 596. Here,cells from cell growth chamber 501 may be harvested by pumping the ICmedia containing the cells through cell harvest path 596 and valve 598to cell harvest bag 599.

Various components of the CES 500 may be contained or housed within amachine or housing, such as cell expansion machine 202 (FIGS. 2 and 3),wherein the machine maintains cells and media, for example, at apredetermined temperature.

In the configuration depicted for CES 500 in FIG. 5A, fluid media infirst fluid circulation path 502 and second fluid circulation path 504flows through cell growth chamber 501 in the same direction (aco-current configuration), in an embodiment. The CES 500 may also beconfigured to flow in a counter-current conformation (not shown), inanother embodiment. In the configuration shown in FIG. 5A, fluid infirst fluid circulation path 502 enters the bioreactor 501 at IC inletport 501A and exits the bioreactor 501 at IC outlet port 501B. In theconfigurations depicted in FIGS. 5B and 5C, fluid media in firstcirculation path 502 may flow in opposite or opposing directions fromconnection 517 such that fluid may enter IC inlet port, a first port,501A on one end of the bioreactor, and fluid may enter IC outlet port, asecond port, 501B on the opposing end of the bioreactor to retain cellsin the bioreactor itself, according to embodiments. The first fluid flowpath may be fluidly associated with the first fluid circulation paththrough connection 517. In embodiments, connection 517 may be a point orlocation from which the fluid may flow in opposite directions, forexample, based on the direction of the IC inlet pump and the directionof the IC circulation pump. In an embodiment, connection 517 may be aT-fitting or T-coupling. In another embodiment, connection 517 may be aY-fitting or Y-coupling. Connection 517 may be any type of fitting,coupling, fusion, pathway, tubing, etc., allowing the first fluid flowpath to be fluidly associated with the first circulation path. It is tobe understood that the schematics and operational configurations shownin FIGS. 5A, 5B, and 5C represent possible configurations for variouselements of the cell expansion system, and modifications to theschematics and operational configurations shown are within the scope ofthe one or more present embodiments.

Turning to FIG. 6, a schematic of another embodiment of a cell expansionsystem 600 is shown. CES 600 includes a first fluid circulation path 602(also referred to as the “intracapillary loop” or “IC loop”) and secondfluid circulation path 604 (also referred to as the “extracapillaryloop” or “EC loop”). First fluid flow path 606 may be fluidly associatedwith cell growth chamber 601 to form first fluid circulation path 602.Fluid flows into cell growth chamber 601 through IC inlet port 601A,through hollow fibers in cell growth chamber 601, and exits via ICoutlet port 601B. Pressure sensor 610 measures the pressure of medialeaving cell growth chamber 601. In addition to pressure, sensor 610may, in embodiments, also be a temperature sensor that detects the mediapressure and temperature during operation. Media flows through ICcirculation pump 612 which may be used to control the rate of mediaflow. IC circulation pump 612 may pump the fluid in a first direction orsecond direction opposite the first direction. Exit port 601B may beused as an inlet in the reverse direction. Media entering the IC loopmay enter through valve 614. As those skilled in the art willappreciate, additional valves, pressure gauges, pressure/temperaturesensors, ports, and/or other devices may be placed at various locationsto isolate and/or measure characteristics of the media along portions ofthe fluid paths. Accordingly, it is to be understood that the schematicshown represents one possible configuration for various elements of theCES 600, and modifications to the schematic shown are within the scopeof the one or more present embodiments.

With regard to the IC loop, samples of media may be obtained from samplecoil 618 during operation. Media then returns to IC inlet port 601A tocomplete fluid circulation path 602. Cells grown/expanded in cell growthchamber 601 may be flushed out of cell growth chamber 601 into harvestbag 699 through valve 698 and line 697. Alternatively, when valve 698 isclosed, the cells may be redistributed within chamber 601 for furthergrowth.

Fluid in second fluid circulation path 604 enters cell growth chamber601 via EC inlet port 601C and leaves cell growth chamber 601 via ECoutlet port 601D. Media in the EC loop may be in contact with theoutside of the hollow fibers in the cell growth chamber 601, therebyallowing diffusion of small molecules into and out of the hollow fibersthat may be within chamber 601, according to an embodiment.

Pressure/temperature sensor 624 disposed in the second fluid circulationpath 604 allows the pressure and temperature of media to be measuredbefore the media enters the EC space of the cell growth chamber 601.Sensor 626 allows the pressure and/or temperature of media in the secondfluid circulation path 604 to be measured after it leaves the cellgrowth chamber 601. With regard to the EC loop, samples of media may beobtained from sample port 630 or a sample coil during operation.

After leaving EC outlet port 601D of cell growth chamber 601, fluid insecond fluid circulation path 604 passes through EC circulation pump 628to oxygenator or gas transfer module 632. EC circulation pump 628 mayalso pump the fluid in opposing directions, according to embodiments.Second fluid flow path 622 may be fluidly associated with oxygenator orgas transfer module 632 via an inlet port 632A and an outlet port 632Bof oxygenator or gas transfer module 632. In operation, fluid mediaflows into oxygenator or gas transfer module 632 via inlet port 632A,and exits oxygenator or gas transfer module 632 via outlet port 632B.Oxygenator or gas transfer module 632 adds oxygen to, and removesbubbles from, media in the CES 600, for example. In various embodiments,media in second fluid circulation path 604 may be in equilibrium withgas entering oxygenator or gas transfer module 632. The oxygenator orgas transfer module 632 may be any appropriately sized device useful foroxygenation or gas transfer. Air or gas flows into oxygenator or gastransfer module 632 via filter 638 and out of oxygenator or gas transferdevice 632 through filter 640. Filters 638 and 640 reduce or preventcontamination of oxygenator or gas transfer module 632 and associatedmedia. Air or gas purged from the CES 600 during portions of a primingsequence may vent to the atmosphere via the oxygenator or gas transfermodule 632.

In the configuration depicted for CES 600, fluid media in first fluidcirculation path 602 and second fluid circulation path 604 flows throughcell growth chamber 601 in the same direction (a co-currentconfiguration). The CES 600 may also be configured to flow in acounter-current conformation, according to embodiments.

In accordance with at least one embodiment, media, including cells (froma source such as a cell container, e.g., a bag) may be attached atattachment point 662, and fluid media from a media source may beattached at attachment point 646. The cells and media may be introducedinto first fluid circulation path 602 via first fluid flow path 606.Attachment point 662 may be fluidly associated with the first fluid flowpath 606 via valve 664, and attachment point 646 may be fluidlyassociated with the first fluid flow path 606 via valve 650. A reagentsource may be fluidly connected to point 644 and be associated withfluid inlet path 642 via valve 648, or second fluid inlet path 674 viavalves 648 and 672.

Air removal chamber (ARC) 656 may be fluidly associated with firstcirculation path 602. The air removal chamber 656 may include one ormore sensors including an upper sensor and lower sensor to detect air, alack of fluid, and/or a gas/fluid interface, e.g., an air/fluidinterface, at certain measuring positions within the air removal chamber656. For example, ultrasonic sensors may be used near the bottom and/ornear the top of the air removal chamber 656 to detect air, fluid, and/oran air/fluid interface at these locations. Embodiments provide for theuse of numerous other types of sensors without departing from the spiritand scope of the present disclosure. For example, optical sensors may beused in accordance with embodiments of the present disclosure. Air orgas purged from the CES 600 during portions of a priming sequence orother protocol(s) may vent to the atmosphere out air valve 660 via line658 that may be fluidly associated with air removal chamber 656.

An EC media source may be attached to EC media attachment point 668, anda wash solution source may be attached to wash solution attachment point666, to add EC media and/or wash solution to either the first or secondfluid flow path. Attachment point 666 may be fluidly associated withvalve 670 that may be fluidly associated with first fluid circulationpath 602 via valve 672 and first fluid inlet path 642. Alternatively,attachment point 666 may be fluidly associated with second fluidcirculation path 604 via second fluid inlet path 674 and second fluidflow path 684 by opening valve 670 and closing valve 672. Likewise,attachment point 668 may be fluidly associated with valve 676 that maybe fluidly associated with first fluid circulation path 602 via firstfluid inlet path 642 and valve 672. Alternatively, fluid container 668may be fluidly associated with second fluid inlet path 674 by openingvalve 676 and closing distribution valve 672.

In the IC loop, fluid may be initially advanced by the IC inlet pump654. In the EC loop, fluid may be initially advanced by the EC inletpump 678. An air detector 680, such as an ultrasonic sensor, may also beassociated with the EC inlet path 684.

In at least one embodiment, first and second fluid circulation paths 602and 604 are connected to waste line 688. When valve 690 is opened, ICmedia may flow through waste line 688 and to waste or outlet bag 686.Likewise, when valve 692 is opened, EC media may flow to waste or outletbag 686.

After cells have been grown in cell growth chamber 601, they may beharvested via cell harvest path 697. Here, cells from cell growthchamber 601 may be harvested by pumping the IC media containing thecells through cell harvest path 697, with valve 698 open, into cellharvest bag 699.

Various components of the CES 600 may be contained or housed within amachine or housing, such as cell expansion machine 202 (FIGS. 2 and 3),wherein the machine maintains cells and media, for example, at apredetermined temperature. It is further noted that, in embodiments,components of CES 600 and CES 500 may be combined. In other embodiments,a CES may include fewer or additional components than those shown in CES500 and/or CES 600 and still be within the scope of the presentdisclosure. An example of a cell expansion system that may incorporatefeatures of the present disclosure is the Quantum® Cell ExpansionSystem, manufactured by Terumo BCT, Inc. in Lakewood, Colo.

It is to be understood that the schematic shown in FIG. 6 represents apossible configuration for various elements of the cell expansionsystem, and modifications to the schematic shown are within the scope ofthe one or more present embodiments.

Examples and further description of cell expansion systems are providedin U.S. Pat. No. 8,309,347 (“Cell Expansion System and Methods of Use,”issued on Nov. 13, 2012) and U.S. Pat. No. 9,057,045, filed on Dec. 15,2010, (“Method of Loading and Distributing Cells in a Bioreactor of aCell Expansion System,” issued on Jun. 16, 2015), which are herebyincorporated by reference herein in their entireties for all that theyteach and for all purposes.

While various example embodiments of a cell expansion system and methodsassociated therewith have been described, FIG. 7 illustrates exampleoperational steps 700 of a process for expanding non-adherent, orsuspension, cells in a cell expansion system, such as CES 500 or CES600, in accordance with embodiments of the present disclosure.

START operation 702 is initiated, and process 700 proceeds topreparation of cells 704. In embodiments, the preparation of cells 704may involve a number of different and optional steps. For example, thecells may be collected 706. The collection of cells 706 may involveseparating and collecting the cells from a donor. In some embodiments,an apheresis procedure may be performed to collect a volume oflymphocytes from the peripheral blood of a donor, e.g., leukapheresis.The volume of lymphocytes may include the target cell population to beexpanded by process 700. In other embodiments, the cells may becollected from cord blood.

After collection 706, optionally, the cells may be isolated 708 as partof the preparation 704. The volume of cells collected at step 706 mayinclude a number of different cell types including the cells that aretargeted for expansion. Optional step 708 may be performed to isolatethe target cells. As one example, the target cells may be T cells, e.g.,regulated T cells. In one embodiment, the regulated T cells may beCD4+CD25+ T cells. The cells may be isolated using any suitableisolation technique. For example, the cells may be isolated usingimmunomagnetic separation where magnetic beads functionalized withantibodies are contacted with the cells collected at 706. Thefunctionalized beads may preferentially attach to the target cellpopulation. A magnetic field may then be used to retain the beads withthe attached target cell population, while the other cells may beremoved.

The cells may be optionally resuspended 710 after isolation 708. Inembodiments, the cells may be resuspended in a media that includes anumber of nutrients and/or reagents that aid in maintaining theviability of the cells. In embodiments, the media may include at leastserum albumin and a reagent, such as a cytokine. The cytokine may inembodiments be a recombinant human IL-2 cytokine. The media may includethe cytokine at a concentrate of 200 IU/ml, in one embodiment.

Following the preparation of the cells 704, process 700 proceeds toexpose cells 712 in order to activate the cells to expand. The cells mayoptionally be exposed to an activator 714 that is soluble. Theactivator, which may include antibody complexes, may be added to themedia in which the cells are resuspended. In embodiments, the activatormay be a human antibody CD3/CD28/CD2 cell activator complex. In someembodiments, the activator, may be included in the media used in theresuspension of the cells 710. Optionally, the cells may be exposed tobeads 716, which may have an activator on their surface. In embodiments,exposing the cells to the beads may involve adding a predeterminedamount of beads to the resuspended cells. The beads may be added atdifferent ratios with respect to the number of cells. For example, thebeads may be added in a 1 bead:2 cell ratio. Other embodiments mayprovide for adding beads at different ratios, e.g., 1 bead:1 cell, 1bead:3 cells, etc. The beads may have antibodies on their surface toactivate the cells to expand. In embodiments, the beads may includeantibodies CD3/CD28 on their surface.

Process 700 proceeds to expand cells 718. As part of cell expansion 718,the cells may be loaded into a cell growth chamber, e.g., a hollow fibermembrane bioreactor, where the cells are expanded. The cells may be fed720 nutrients to promote their expansion. For example, media may bedelivered into the cell growth chamber to provide the nutrients forexpansion. The expansion of the cells 718 may also include addingreagents periodically to the cell growth chamber to continue to promotetheir expansion. For example, in some embodiments, reagents (e.g.,cytokines) may be added to the cell growth chamber to promote theexpansion of the cells. In one embodiment, the reagent may be additionalIL-2 cytokine, e.g., recombinant human IL-2 cytokine.

Also as part of expanding the cells 718, the environment inside the cellgrowth chamber may be controlled. For example, gasses may be deliveredand exchanged continuously to provide a balance of for example, carbondioxide and oxygen to the cells expanding in the cell growth chamber.Additionally, the temperature may be controlled to be within a rangeoptimized for cell expansion. Expansion of cells 718 may also includemonitoring metabolites 726. For example, the lactate and glucose levelsmay be periodically monitored. A rise or fall in the metabolites mayprompt changes (e.g., additional feeding, additional reagent additions,additional gas exchange, etc.) to control the environment 724 in thecell growth chamber.

Process 700 next proceeds to harvest the cells 728. Further processingof the removed cells or other analysis may optionally be performed atstep 730. For example, the cells may be characterized to determine cellphenotype(s). The further processing or other analysis 730 may includeperforming flow cytometry, for example, to characterize cell phenotypes.Process 700 may then terminate at END operation 732. If it is notdesired to perform further processing/analysis, process 700 terminatesat END operation 732.

FIG. 8A illustrates operational steps 800 of a process, which may beused to position cells or other material (e.g., proteins, nutrients,growth factors) into a cell growth chamber according to embodiments ofthe present disclosure. In embodiments, the process 800 may beimplemented as part of a “load cells centrally without circulation”task. Start operation 802 is initiated and process 800 proceeds to step804, where a first volume of fluid with cells may be loaded into a cellgrowth chamber of a cell expansion system. In embodiments, the cells maycomprise non-adherent cells, such as one or more types of T cells. Inone embodiment, the plurality of cells comprises Tregs. As may beappreciated, loading of the first volume of fluid with cells may beperformed by components of a cell expansion system such as systems CES500 (e.g., FIG. 5A) and CES 600 (FIG. 6), described above. FIG. 8Billustrates a portion of a cell expansion system that includes a firstinlet pump 840, a first fluid flow path 860, a first fluid circulationpump 848, a first fluid circulation path 852, a cell growth chamber 844,and a second fluid circulation path 854. The first fluid flow path 860is fluidly associated with fluid circulation path 852 through connection860B. Embodiments may provide for the first volume of fluid with thecells to be loaded 804 through the first fluid flow path 860 utilizingthe first fluid inlet pump 840 and into the first fluid circulation path852. In embodiments, the first volume of fluid is loaded withoutactivating the first fluid circulation pump 848.

As illustrated in FIG. 8B, a volume of the first fluid circulation path852 may be comprised of a number of volumes of its portions. Forexample, a first portion of the volume may be an intracapillary space(when the cell growth chamber is a hollow fiber membrane bioreactor) ofthe cell growth chamber 844. A second portion of the volume may be fromconnection 860B to an inlet port 844A of the cell growth chamber 844. Athird portion may be from the connection 860B to an outlet port 844A ofthe cell growth chamber 844.

Process 800 proceeds to loading of a second volume of fluid 806. Thesecond volume of fluid may comprise media and may be introduced into aportion of the first fluid flow path 860. In embodiments, the secondvolume may be a predetermined amount selected in order to position 808the first volume into a first portion of the cell growth chamber 844. Inembodiments, the first volume of fluid and the second volume of fluidmay be the same. In other embodiments, the first volume of fluid and thesecond volume of fluid may be different. In yet other embodiments, a sumof the first volume of fluid and the second volume of fluid may be equalto a percentage of a volume of the first fluid circulation path, e.g.,path 852 (FIG. 8B).

In order to position the first volume of fluid, the second volume offluid has to be enough to push the first volume into the desiredposition in the cell growth chamber 844. The second volume of fluid maytherefore, in embodiments, be about as large as the volume of the firstfluid circulation path 852 between connection 860B and the inlet port844A. As may be appreciated, this would push the first volume of fluidwith the cells into a position within the cell growth chamber 844.

In other embodiments, the first volume of fluid (with cells) may bepositioned 808 about a central region 866 of the cell growth chamber844. In these embodiments, the second volume may be about as large a sumof the volume of the first fluid circulation path 852, betweenconnection 860B and the inlet port 844A and the volume of the firstfluid circulation path made up by the cell growth chamber 844 (e.g., thevolume of the intracapillary space) that would not be occupied by thefirst volume of fluid when positioned in the cell growth chamber. Forexample, in one embodiment, the first volume of fluid (with the cells)may be 50 ml. The cell growth chamber may have a volume of, for example,124 ml. When the first volume is positioned around the central region866, it will occupy the 50 ml around the central region 866, leaving 74ml, which is on either side of the central region 866. Accordingly, 50%of 74 ml (or 37 ml) may be added to the volume between connection 860and inlet port 844A to position the 50 ml of the first volume around thecentral region 866.

Positioning 808 the first volume may in embodiments, involve addingadditional volumes of fluid to position the first volume with cells inthe cell growth chamber. For example, if the desired position of thefirst volume is not achieved with the second volume, additional fluidmay be added to position the first volume 808.

Process 800 proceeds to the query 810 to determine whether the firstvolume should be repositioned. For example, in embodiments, the firstvolume may be positioned closer to inlet port 844A. If it is desired tomove the first volume closer to central region 866 of cell growthchamber 844, process 800 may loop back to step 808 where additionalfluid may be added to the first fluid circulation path to position thefirst volume of fluid.

If it is determined at query 810 that the first volume does not need tobe repositioned, process 800 proceeds to feeding of the cells 812. Inembodiments, the cells may be fed using a media that includes a numberof compounds such as glucose, proteins, growth factors, reagents, orother nutrients. In embodiments, feeding of the cells 812 may involveactivating inlet pumps and circulation pumps (e.g., pumps 840 and 848)to deliver media with nutrients to the cells in the cell growth chamber844. Some embodiments provide for the cells to be maintained in the cellgrowth chamber 844 during step 812, as described below. Theseembodiments may involve activating pumps, e.g., inlet pumps andcirculation pumps (e.g., pumps 840 and 848, as described below) so thatflow of fluid into the cell growth chamber 844 may be from bothdirections such as from the inlet port 844A and the outlet port 844Binto the cell growth chamber 844.

Process 800 then proceeds to the expansion of the cells 814 where thecells may be expanded or grown. While step 814 is shown after step 812,step 814 may occur before, or simultaneous with, step 812, according toembodiments. Cells may then be removed 816 from the cell growth chamberand collected in a storage container. In embodiments, step 816 mayinvolve a number of sub-steps. For example, the cells may be circulatedby the circulation pump (e.g., pump 848) before they are collected andstored in a container. Process 800 terminates at END operation 830.

Next, FIG. 9A illustrates example operational steps 900 of a process forretaining cells in a bioreactor of a cell expansion system, such as CES500 (e.g., FIGS. 5B & 5C), in accordance with embodiments of the presentdisclosure. As discussed above, cells residing in the headers of thebioreactor or in the IC loop outside of the bioreactor may not receiveproper gas exchange and nutrient exchange, which may result in cellaggregation and death. In an embodiment, the bioreactor provides gasexchange and nutrient exchange through the semi-permeable hollow fibermembrane. It can be important that such exchange is efficient as thesurface area to volume ratio in a cell expansion system comprising ahollow fiber membrane may be significantly larger than that of othercell culturing methods (e.g., about 15 times that of cell cultureflasks, for example). Such efficiency may be accomplished by minimizingthe diffusion distance for media constituents able to pass the membranesurface where exchange takes place.

For example, FIG. 9B illustrates a graph 936 of the oxygen consumptiondemands placed on a cell expansion system, such as the Quantum® CellExpansion System during cell proliferation (approximately 3E+09 T-cellspresent in the bioreactor). FIG. 9B depicts the percentage (%) of oxygen(O₂) at the bioreactor outlet 938. For example, a sensor to measureoxygen levels may be placed at the EC outlet port of the bioreactor,according to an embodiment. In another embodiment, a sensor to measureoxygenation may be placed at the IC outlet port of the bioreactor. Thepercentage of O₂ is measured against the Run Time (e.g., in minutes)940. To maximize the oxygen supply to the cells, the EC circulation flowrate, Q_(EC Circ) may be set to 300 mL/min (942), according to anembodiment. FIG. 9B shows a change in oxygenation when the ECcirculation rate is dropped from 300 mL/min (942) to 50 mL/min (944).For example, the cells may consume oxygen (O₂) in the media as the mediatravels across the bioreactor. Fluid at 50 mL/min (944) moves moreslowly across the bioreactor than fluid at 300 mL/min (942), so theremay be a longer time period or greater opportunity for the cells tostrip the media of oxygen when the EC circulation rate is at 50 mL/min.The oxygenation then recovers when the EC circulation rate is taken backup to 300 mL/min (946). FIG. 9B shows a possible benefit of keeping thecells in the fibers themselves where gas transfer takes place, asopposed to in the portion of the IC circulation path outside of thebioreactor, for example, where the cells may be deprived of oxygen. Itmay therefore be beneficial to retain cell, e.g., non-adherent cell,populations inside the hollow fibers of the bioreactor during feeding bydirecting the flow of media to enter both sides of the bioreactor, e.g.,the IC inlet port and the IC outlet port. In an embodiment, an equaldistribution of flow to the IC inlet port and the IC outlet port may beused. In another embodiment, a larger flow to the IC inlet port ascompared to the IC outlet port may be used and vice versa, depending onwhere it is desired to locate the cells in the bioreactor, for example.

Returning to FIG. 9A, START operation 902 is initiated and process 900proceeds to load a disposable set or premounted fluid conveyanceassembly (e.g., 210 or 400) onto a cell expansion system 904. Thedisposable set may then be primed 906, in which the set may be primed906 with Lonza Ca²⁺/Mg²⁺-free PBS, for example. In preparation for theloading and seeding of cells, the priming fluid may be exchanged usingan IC/EC washout 908. In an embodiment, the PBS in the system may beexchanged for TexMACS GMP Base Medium, for example. Next, process 900proceeds to close the IC outlet valve 910. In embodiments, the EC outletvalve may be open to allow for ultrafiltration of fluid added to thehollow fibers of a bioreactor comprising a hollow fiber membrane. Themedia may next be conditioned 912. Next process 900 proceeds to loadcells 914, e.g., suspension or non-adherent cells, such as T cells orTregs. In an embodiment, such cells may be loaded 914 by a “load cellscentrally without circulation” task. In another embodiment, such cellsmay be loaded 914 by a “load cells with uniform suspension” task. Inother embodiments, other loading tasks and/or loading procedures may beused.

In an embodiment, the cells being loaded 914 may be suspended in asolution comprising media to feed the cells during and after suchloading, for example. In another embodiment, such solution may compriseboth media for feeding the cells and a soluble activator complex tostimulate the cells, e.g., T cells. Such loading 914 may occur on Day 0,for example.

Following the loading of cells 914, the cells may be further fed 916.During such feeding 916, it may be desired to control the cell residencein the bioreactor itself. Through the adjustment of flow controlparameters 918, the cells may be retained in the bioreactor itselfinstead of losing cells from the bioreactor into the portion(s) of theIC loop outside of the bioreactor, for example, during the expansionphase of growth. By retaining the cells in the bioreactor, cells in thebioreactor may be closer to the IC inlet port, in which such cells mayreceive the freshest growth media, according to embodiments. On theother hand, cells in the IC loop may be receiving expended orconditioned media which may affect their glycolytic metabolism, forexample. In addition, cells in the bioreactor may receive mixed gas(e.g., oxygen, carbon dioxide, and nitrogen) input from a gas transfermodule (GTM) by diffusion from the EC loop circulation, whereas cells inother portions of the IC loop may not receive such mixed gas, accordingto an embodiment. It should be noted that while embodiments may providefor retaining the cells in the bioreactor itself, other embodiments mayprovide for maintaining the cells in any location allowing for improvednutrient delivery and/or gas exchange. Embodiments thus provide for theuse of other locations for retaining cells, or controlling the residenceof cells, without departing from the spirit and scope of the presentdisclosure.

Returning to FIG. 9A and process 900, the loss of cells from the hollowfiber membrane bioreactor may be reduced by matching, or closely orsubstantially matching, the IC circulation pump rate to the IC inletpump rate, but in the opposite direction, in accordance withembodiments. The IC inlet pump 920 may be adjusted to produce a firstflow rate or volumetric flow rate, and the IC circulation pump may beadjusted 922 to produce a second counter-flow rate or secondcounter-volumetric flow rate, in which a volumetric flow rate or fluidflow rate or rate of fluid flow or flow rate may be considered as thevolume of fluid which passes per unit time (may be represented by thesymbol “Q”). For example, an IC inlet pump rate of 0.1 mL/min may bematched, or closely or substantially matched, to a complementary ICcirculation pump rate of −0.1 mL/min to maintain cells in the bioreactorduring the growth phase of the cell culture, which may be Days 4-7, forexample, in embodiments. Such pump adjustment 918 may allow forcounteracting any forces associated with a loss of cells from the ICoutlet port of the bioreactor, for example.

Next, the cells may be allowed to grow or expand 924. The cells are notlimited to growing or expanding at step 924, but, instead, the cells mayalso expand during step(s) 914, 916, 918, 920, 922, for example. Process900 may next proceed to harvest operation 926, in which the cells may betransferred to a harvest bag(s) or container(s). The disposable set maythen be unloaded 932 from the cell expansion system, and process 900then terminates at END operation 934.

Alternatively, from harvest operation 926, process 900 may optionallyproceed to allow for further processing/analysis 928. Such furtherprocessing 928 may include characterization of the phenotype(s), forexample, of the harvested cells. From optional furtherprocessing/analysis step 928, process 900 may proceed to optionallyreload any remaining cells 930. Process 900 may then proceed to unloadthe disposable set 932, and process 900 may then terminate at ENDoperation 934. Alternatively, process 900 may proceed from furtherprocessing/analysis step 928 to unload disposable set 932. Process 900may then terminate at END operation 934.

Next, FIG. 10A illustrates example operational steps 1000 of a processfor feeding cells that may be used with a cell expansion system, such asCES 500 (e.g., FIGS. 5B &5C), in accordance with embodiments of thepresent disclosure. START operation 1002 is initiated, and process 1000proceeds to load a disposable set onto the cell expansion system, primethe set, perform an IC/EC washout, condition media, and load cells,e.g., suspension or non-adherent cells, for example. Next, process 1000proceeds to feed the cells during a first time period 1004. Inembodiments, a first inlet flow rate and a first circulation flow ratemay be used. As an example, a first IC inlet flow rate and a first ICcirculation flow rate may be used, in which the first IC inlet flow ratemay be controlled by the IC inlet pump, e.g., first pump, and the firstIC circulation flow rate may be controlled by the IC circulation pump,e.g., second pump. In an example embodiment, the IC inlet pump (554) maycause a volumetric flow rate of 0.1 mL/min to enter the IC inlet port(501A) of the bioreactor (501) with the IC circulation pump (512)causing a complementary IC circulation volumetric flow rate or fluidflow rate of −0.1 mL/min to enter the IC outlet port (501B) of thebioreactor (501), in which the negative symbol (“−”) used in −0.1mL/min, for example, indicates a direction of the IC circulation pump(512) to cause or produce a counter-flow rate to maintain cells in thebioreactor during the growth phase of the cell culture.

During the feeding of the cells and the use of the IC pumps duringfeeding to control cell residence in the bioreactor through flow andcounter-flow properties, the cells continue to grow and expand. As aresult, the cells may demand additional media, e.g., glucose and/or cellgrowth formulated media, to support the expanding population. Effortsmay also be made to control lactate values of the expanding cellpopulation. In embodiments, cell culture lactate values may bemaintained at or below about 20 mmol/L, at or below about 15 mmol/L, ator below about 10 mmol/L, or even at or below about 7 mmol/L. In otherembodiments, rate(s) of media addition, for example, and/or othersettings may be controlled to attempt to maintain the lactatelevels≤about 5 mmol/L, for example, to improve cell growth andviability. Other concentrations may be used in other embodiments.

In an example embodiment, an effort may be made to control lactatevalues at about 7 mmol/L by concurrently increasing both the IC inlet(+) pump rate and IC circulation (−) pump rate from ±0.1 to ±0.4 mL/minwithin the lumen of the hollow fiber membrane over multiple timeperiods, e.g., days (Days 4-8), according to embodiments. For example,FIG. 10B provides a table 1018 of example IC pump rates for feedingusing a “feed cells” task, for example, with a cell expansion system(e.g., CES 500). Table 1018 provides example time periods 1020, e.g.,Days, versus example IC pump rates 1022 to produce volumetric flow ratesto both sides of the bioreactor to maintain cells in the bioreactor. Forexample, Days 0-4 (1024) may use an IC inlet or input pump rate of 0.1mL/min (1026) and an IC circulation pump rate of −0.1 mL/min (1028); Day5 (1030) may use an IC inlet pump rate of 0.2 mL/min (1032) and an ICcirculation pump rate of −0.2 mL/min (1034); Day 6 (1036) may use an ICinlet pump rate of 0.3 mL/min (1038) and an IC circulation pump rate of−0.3 mL/min (1040); and Day 7 (1042) may use an IC inlet pump rate of0.4 mL/min (1044) and an IC circulation pump rate of −0.4 mL/min (1046).While table 1018 of FIG. 10B provides example pump rates of ±0.1 to ±0.4mL/min for feeding the cells while retaining the cells in the bioreactorduring the growth phase of the cell culture, other pump rates andresulting flow rates may be used according to embodiments withoutdeparting from the spirit and scope of the present disclosure. Forexample, while increments of ±0.1 mL/min for increasing the feed flowrate are shown in this example, other increments, e.g., ±0.005 mL/min,±0.05 mL/min, etc., may be used to increase the feed flow rate inembodiments. The time periods, e.g., Days, and pump rates in table 1018of FIG. 10B are offered merely for illustrative purposes and are notintended to be limiting.

Returning to FIG. 10A, process 1000 proceeds from feeding the cellsduring a first time period 1004 to increasing the first inlet flow rateby a first amount to achieve a second inlet flow rate 1006. For example,as in the embodiment depicted in FIG. 10B as discussed above, the ICinlet pump rate (+) may increase from 0.1 mL/min to 0.2 mL/min toproduce an IC inlet flow rate of 0.2 mL/min. Further, the IC circulationpump rate (−) may concurrently increase from −0.1 mL/min to −0.2 mL/minto produce an IC circulation flow rate of −0.2 mL/min. The firstcirculation flow rate is thus increased by the first amount to achievethe second circulation flow rate 1008. The cells may then be fed duringa second time period at the second inlet flow rate and at the secondcirculation flow rate 1010 to maintain the cells in the bioreactor andoutside of the headers and outside of the portion of the IC circulationpath outside of the bioreactor, for example. Following the second timeperiod of feeding 1010, process 1000 may terminate at END operation 1016if it is not desired to continue feeding and/or expanding the cells, forexample. Alternatively, process 1000 may optionally continue toincrease, or otherwise change, the feeding flow rates 1012. There may beany number of feeding time periods, as represented by ellipsis 1014.Following the desired number of feeding time periods 1014, process 1000then terminates at END operation 1016. While FIGS. 10A and 10B andprocess 1000 show “increases” in the flow rates, other adjustments tothe flow rates may be made. For example, the flow rates may decrease orremain substantially the same from one feeding period to the next.Considerations, such as metabolic activity, for example, may determinehow flow rates are adjusted. The “increases” in flow rates in FIGS. 10Aand 10B are offered merely for illustrative purposes and are notintended to be limiting.

Turning to FIG. 11A, example operational steps 1100 of a process forfeeding cells that may be used with a cell expansion system, such as CES500 (e.g., FIGS. 5B & 5C), are provided in accordance with embodimentsof the present disclosure. START operation is initiated 1102, andprocess 1100 proceeds to load a disposable set onto the cell expansionsystem, prime the set, perform an IC/EC washout, condition media, andload cells, e.g., suspension or non-adherent cells, for example. Next,process 1100 proceeds to feed the cells during a first time period 1104.In embodiments, a first inlet rate or flow rate and a first circulationrate or flow rate may be used. As an example, a first IC inlet flow rateand a first IC circulation flow rate may be used, in which the first ICinlet flow rate may be controlled by the IC inlet pump, e.g., firstpump, and the first IC circulation flow rate may be controlled by the ICcirculation pump, e.g., second pump. In an example embodiment, the ICinlet pump (554) may cause a volumetric flow rate or fluid flow rate of0.1 mL/min to enter the IC inlet port (501A) of the bioreactor (501)with the IC circulation pump (512) causing a complementary ICcirculation flow rate of −0.1 mL/min to enter the IC outlet port (501B)of the bioreactor (501), in which the negative symbol (“−”) used in −0.1mL/min, for example, indicates a direction of the IC circulation pump(512) to cause or produce a counter-flow rate to maintain cells in thebioreactor during the growth phase of the cell culture.

During the feeding of the cells and the use of the IC pumps to controlcell residence in the bioreactor through flow and counter-flowproperties, the cells continue to grow and expand. As a result, thecells may demand additional media, e.g., glucose and/or cell growthformulated media, to support the expanding population. Efforts may alsobe made to control lactate values of the expanding cell population. Inan example embodiment, an effort may be made to control lactate valuesat about 7 mmol/L, for example, by concurrently increasing both the ICinlet (+) pump rate and IC circulation (−) pump rate from ±0.1 to ±0.4mL/min within the lumen of the hollow fiber membrane over multiple days,e.g., Days 4-8, according to embodiments. For example, see FIG. 10B,table 1018, and the discussion above, for example pump rates forfeeding. As noted, while table 1018 of FIG. 10B provides pump rates of±0.1 to ±0.4 mL/min for feeding the cells while retaining the cells inthe bioreactor during the growth phase of cell culturing, other pumprates and resulting flow rates may be used according to embodimentswithout departing from the spirit and scope of the present disclosure.The time periods, e.g., Days, and pump rates in table 1018 of FIG. 10Bare offered merely for illustrative purposes and are not intended to belimiting.

Returning to FIG. 11A, process 1100 proceeds from feeding the cellsduring a first time period 1104 to increasing the first inlet rate orflow rate by a first amount to achieve a second inlet rate or flow rate1106. For example, as in the embodiment depicted in FIG. 10B asdiscussed above, the IC inlet pump rate (+) may increase from 0.1 mL/minto 0.2 mL/min to produce an IC inlet flow rate of 0.2 mL/min. Further,the IC circulation pump rate (−) may concurrently increase from −0.1mL/min to −0.2 mL/min to produce an IC circulation flow rate of −0.2mL/min. The first circulation rate or flow rate is thus increased by thefirst amount to achieve the second circulation rate or flow rate 1108.The cells may then be fed during a second time period at the secondinlet rate or flow rate and at the second circulation rate or flow rate1110 to maintain the cells in the bioreactor and outside of the headersor the portion of the IC circulation path outside of the bioreactor, forexample. Following the second period of feeding 1110, process 1100 mayterminate at END operation 1116 if it is not desired to continue feedingand/or expanding the cells, for example.

Alternatively, process 1100 may optionally determine whether to adjustthe feeding rates or flow rates based on metabolic activity, in whichprocess 1100 proceeds to optional query 1112 to determine whether toadjust feeding based on metabolic levels. Monitoring the glucose and/orlactate levels can facilitate the adjustment of cell expansion systemmedia flow rates, e.g., IC media flow rate, to support cell, e.g., Treg,expansion in a bioreactor, such as a hollow fiber bioreactor, forexample.

As shown in FIGS. 11B and 11C, embodiments provide for controlling thelactate values of cell expansion runs or procedures involving theexpansion of hTregs, for example. Graphs 1118 and 1132 indicate thatmeasurements of glucose and lactate levels may be used to adjust cellexpansion system media flow rates, e.g., IC media flow rates, to supportthe expansion of Tregs, for example. For example, FIG. 11B provides agraph 1118 showing the metabolisms of expanding hTregs, in which suchcell expansion may occur in a cell expansion system, such as theQuantum® Cell Expansion System. The glucose concentration (mg/dL) 1120and lactate concentration (mmol/L) 1122 are shown for various cellexpansion runs 1124 and 1125 across periods of time, e.g., Days, 1126.Throughout these Treg runs 1124 and 1125, an effort may be made tocontrol the lactate values of the expanding cell population to valuesabout 7 mmol/L by concurrently increasing both the IC inlet (+) pumprate and IC circulation pump rate from ±0.1 to ±0.4 mL/min, for example,within the lumen of the hollow fiber membrane over Days 4-8, forexample. In other embodiments, other pump rates may be used. As shown,the lowest glucose levels during the Treg cell expansions may range froma concentration 1128 of 264 mg/dL on Day 7 (Q1584) to a concentration1130 of 279 mg/dL on Day 8 (Q1558), according to the embodiments shown.As depicted, the base glucose concentration, in the cell growthformulated medium for runs 1124 may range from 325 mg/dL to 335 mg/dL,for example. In other embodiments, it may be desired to maintain thelactate levels≤about 5 mmol/L to improve cell growth and viability. Inembodiments, graphical user interface (GUI) elements may be used tocontrol the rate of media addition and to maintain the lactate metabolicwaste product from glycolysis below a defined level during the expansionof cells.

Turning to FIG. 11C, graph 1132 shows the metabolisms of expandinghTregs, in which such cell expansion may occur in a cell expansionsystem, such as the Quantum® Cell Expansion System. The glucoseconsumption (mmol/day) 1134 and lactate generation (mmol/day) 1136 areshown for various cell expansion runs 1138 and 1139 across periods oftime 1140, e.g., Days. To control lactate values at about 7 mmol/L, forexample, concurrent increases may be made to the IC inlet (+) pump rateand IC circulation (−) pump rates from ±0.1 to ±0.4 mL/min, in anembodiment. For example, graph 1132 shows IC circulation and IC feedrates of ±0.1 mL/min (1142); ±0.2 mL/min (1144); ±0.3 mL/min (1146); and±0.4 mL/min (1148). Other embodiments may use other flow rates. The flowrates used and shown in FIGS. 11B and 11C are offered for purposes ofillustration and are not intended to be limiting. It is noted that whilepositive (+) may be shown for the direction of the IC inlet pump andnegative (−) may be shown for the direction of the IC circulation pump,such directions are offered for illustrative purposes only, in whichsuch directions depend on the configurations of the pumps used.

Returning to FIG. 11A and optional query 1112, if it is not desired tomeasure metabolic activity and/or adjust feeding levels based on suchmeasurement(s), process 1100 proceeds “no” to END operation 1116, andprocess 1100 terminates. Alternatively, where it is desired to adjustfeeding levels based on metabolic activity, process 1100 proceeds “yes”to optional step 1114 to continue to increase the rate of media additionand feed the expanding cell population. While step 1114 is shown as onestep, this step may involve numerous adjustments to the media additionrate, such as increasing the IC inlet flow rate and increasing the ICcirculation flow rate, for example. Step 1114 is shown as one step onlyfor illustrative purposes and is not intended to be limiting. Followingany adjustments to the rate of media addition, process 1100 proceeds tooptional query 1112 to determine whether to continue measuring metaboliclevels and/or adjust feeding. If it is not desired to continue measuringmetabolic activity and/or adjusting feeding levels based on metabolicactivity, process 1100 proceeds “no” to END operation 1116, and process1100 terminates. While FIG. 11A and process 1100 show “increases” in therates or flow rates, other adjustments to the flow rates may be made.For example, the rates or flow rates may decrease or remainsubstantially the same from one feeding period to the next. The type ofadjustments which may be made may depend on the metabolic activityassessment of the growing cell population. The “increases” in flow ratein FIG. 11A, for example, are offered merely for illustrative purposesand are not intended to be limiting.

Next, FIG. 12 illustrates example operational steps 1200 of a processfor retaining cells in a location during feeding using a cell expansionsystem, such as CES 500 (e.g., FIGS. 5B &5C), in accordance withembodiments of the present disclosure. START operation 1202 isinitiated, and process 1200 proceeds to load a disposable set onto thecell expansion system, prime the set, perform IC/EC washout, conditionmedia, and load cells, e.g., suspension or non-adherent cells, forexample. Next, process 1200 proceeds to feed the cells during a firsttime period 1204. In embodiments, a first inlet flow rate and a firstcirculation flow rate may be used. As an example, a first IC inlet flowrate and a first IC circulation flow rate may be used, in which thefirst IC inlet flow rate may be produced and controlled by the IC inletpump, e.g., first pump, and the first IC circulation flow rate may beproduced and controlled by the IC circulation pump, e.g., second pump.In an example embodiment, the first IC inlet pump (554) may cause avolumetric flow rate of 0.1 mL/min to enter the IC inlet port (501A) ofthe bioreactor with the IC circulation pump (512) causing acomplementary IC circulation volumetric flow rate or fluid flow rate of−0.1 mL/min to enter the IC outlet port (501B) of the bioreactor (501),in which the negative symbol (“−”) used in −0.1 mL/min, for example,indicates a direction of the IC circulation pump to cause or produce acounter-flow rate to maintain cells in the bioreactor during the growthphase of the cell culture. In another embodiment, the first ICcirculation flow rate may be a percentage of the first IC inlet flowrate. For example, the first IC circulation flow rate may be about fiftypercent (50%) or about one-half (%), or another percentage or portionaccording to embodiments, of the first IC inlet flow rate.

During the feeding (and expansion) of the cells and the use of the ICpumps, for example, to control cell residence in the bioreactor throughflow and counter-flow properties, the cells continue to grow and expand.As a result, the cells may demand additional media, e.g., glucose and/orcell growth formulated media, to support the expanding population. Inembodiments, efforts may be made to increase the rate of media additionto feed the expanding cell population. In an example embodiment, anincrease in the IC inlet pump rate (+) may cause the IC inlet flow rateto increase by a first amount to achieve a second IC inlet flow rate1206. For example, the IC inlet flow rate may increase by a first amountof 0.1 mL/min to achieve a second IC inlet flow rate of 0.2 mL/min,according to an embodiment. Other adjustments may be made to the ICinlet flow rate according to other embodiments.

Next, the second IC circulation flow rate may be set, or adjusted orconfigured, to equal a percentage, or portion, or fraction of the secondIC inlet flow rate 1208, according to embodiments. For example, thesecond IC circulation flow rate may be set, or adjusted or configured,to equal about fifty percent (50%) or about one-half (½), or anotherpercentage or portion according to embodiments, of the value of the ICinlet flow rate, according to an embodiment. Depending on the value ofthe first IC circulation flow rate, an adjustment to the IC circulationpump rate (−) may cause the second IC circulation flow rate to increase,according to an embodiment. In another embodiment, an adjustment may bemade to the IC circulation pump rate to produce or cause the second ICcirculation flow rate to decrease such that the IC circulation flow ratemay be substantially equal to a pre-defined percentage or pre-definedfraction of the second IC inlet flow rate. In yet another embodiment, noadjustment may be made to the IC circulation pump rate. For example,where the first IC inlet flow rate equals 0.1 mL/min, and the first ICcirculation flow rate equals −0.1 mL/min, if the second IC inlet flowrate is increased to 0.2 mL/min, the second IC circulation flow rate maybe set to (−½)*(second Q_(IC Inlet)) (where Q_(IC Inlet) is the IC inletflow rate) or (−½)*(0.2 mL/min), which provides for a second Q_(IC Circ)(where Q_(IC Circ) is the IC circulation flow rate) of −0.1 mL/min, andno adjustment to the IC circulation pump rate may be made to achievesuch second Q_(IC Circ).

Turning to FIGS. 5B and 5C, such figures depict operationalconfigurations of cell expansion system 500 showing fluid movement inthe first circulation path, in accordance with embodiments of thepresent disclosure. The configurations of FIGS. 5B and 5C show a split,for example, in the flow rate to the IC inlet port, e.g., first port,and to the IC outlet port, e.g., second port, to keep the cells in thecell growth chamber or bioreactor. As discussed with respect to CES 500above, in the IC loop or first circulation path 502, fluid may beinitially advanced by the IC inlet pump 554. Such fluid may be advancedin a first direction, such as a positive direction, for example. Fluidmay flow into cell growth chamber or bioreactor 501 through IC inletport 501A, through hollow fibers in cell growth chamber or bioreactor501, and may exit via IC outlet port 501B. Media may flow through ICcirculation pump 512 which may be used to control the rate of mediaflow. IC circulation pump may pump the fluid in a first direction orsecond direction opposite the first direction. Exit port 501B may beused as an inlet in the reverse direction, for example. In anembodiment, the IC circulation pump 512 may pump the fluid in adirection opposite the direction of the IC inlet pump, for example. Asan example, the direction of the IC inlet pump may be positive (+), andthe direction of the IC circulation pump may be negative (−) to cause acounter-flow such that fluid may enter both sides of the bioreactor tokeep the cells in the bioreactor.

In an embodiment, a first portion of fluid may branch at connection 517to flow into the IC inlet port (501A) of the bioreactor 501. In anembodiment, the IC circulation pump 512 may operate at a pump rate thatmay be matched, or closely or substantially be matched, to the IC inletpump 554 rate, but in the opposite direction, such that a second portionof fluid may branch at connection 517 to flow into the IC outlet port(501B) of the bioreactor 501. For example, an IC inlet pump rate of +0.1mL/min may be matched, or closely or substantially be matched, to acomplementary IC circulation pump rate of −0.1 mL/min to maintain cellsin the bioreactor during the growth phase of the cell culture. Such pumpadjustment tactic during the feeding may counteract forces associatedwith the loss of cells from the IC outlet port. Such type of feedingusing pump adjustments to cause flow and counter-flow into the IC inletport (501A) and IC outlet port (501B), respectively, of the bioreactormay be referred to as a modified feeding method, according to anembodiment. In another example embodiment, the IC circulation pump ratemay be adjusted to cause a flow rate into the IC outlet port (501B) thatmay be equal to about fifty percent (50%) or about one-half (½), oranother percentage or fraction in other embodiments, of the IC inletflow rate, but in the opposite direction. For example, with an IC inletpump rate of 0.4 mL/min, an IC circulation pump rate may be set, orconfigured or adjusted, to about −0.2 mL/min. Other percentages orportions may be used in other embodiments.

FIGS. 5B and 5C illustrate operational configurations showing fluidmovement in CES 500, in which the flow and counter-flow rates to keepthe cells in the cell growth chamber or bioreactor 501 are shown,according to embodiments. For example, such flow rates are illustratedin FIG. 5B as “X” flow rate 503; “(−½) X” flow rate 505 (where thenegative symbol (“−”) is an indication of direction, in which adirection of flow rate 505 is shown by the directional arrow in FIG.5B); and (½) X″ flow rate 507, in which about ½, or a first portion, ofthe flow rate branches at connection 517 to enter IC inlet port (501A)of bioreactor 501, and about ½, or a second portion, of the flow ratebranches at connection 517 to enter the IC outlet port (501B) ofbioreactor 501, according to embodiments. As shown, the sum of the firstportion and the second portion may be substantially equal to the totalflow rate 503, in which the total flow rate 503 is pumped by IC inletpump 554. Depending on the flow and counter-flow rates which may be usedto keep the cells in the bioreactor or cell growth chamber 501, othertypes of fractions or percentages of the IC inlet pump rate may be usedto set, or configure or adjust, the IC circulation pump. As such, FIG.5C illustrates such flow rates as “X” flow rate 511; “−(y %)*X” flowrate 513 (where the negative symbol (“−”) is an indication of direction,in which a direction of flow rate 513 is shown by the directional arrowin FIG. 5C); and “(100%−y %)*X” flow rate 515, where “y” equals anumeric percentage, according to embodiments. In embodiments, the sum offlow rate 513 and flow rate 515 is substantially equal to flow rate 511.

In an embodiment, all, or substantially all, of the flow from the firstfluid flow path 506 may flow to IC inlet port (501A) of bioreactor 501from connection 517, for example. In another embodiment, all, orsubstantially all, of the flow from the first fluid flow path 506 mayflow to IC outlet port (501B) of bioreactor 501 from connection 517. Inyet another embodiment, a first portion of the flow from first fluidflow path 506 may flow from connection 517 to IC inlet port (501A), anda second portion of the flow from the first fluid flow path 506 may flowfrom connection 517 to IC outlet port (501B). In embodiments, thepercentage of the IC inlet flow rate at which the IC circulation flowrate may be set may range from about 0 percent to about 100 percent. Inother embodiments, the percentage may be between about 25 percent andabout 75 percent. In other embodiments, the percentage may be betweenabout 40 percent and about 60 percent. In other embodiments, thepercentage may be between about 45 percent and about 55 percent. Inembodiments, the percentage may be about 50 percent. It is to beunderstood that the operational configurations shown in FIGS. 5B and 5Crepresent possible configurations for various operations of the cellexpansion system, and modifications to the configurations shown arewithin the scope of the one or more present embodiments.

Returning to FIG. 12, the cells may be fed (and continue expanding)during a second time period at the second inlet flow rate and at thesecond circulation flow rate 1210 to maintain the cells in thebioreactor and outside of the headers or portion of the IC circulationpath outside of the bioreactor, for example. Following the second periodof feeding 1210, process 1200 may terminate at END operation 1216 if itis not desired to continue feeding and/or expanding the cells, forexample. Alternatively, process 1200 may optionally continue toincrease, or otherwise change, the feeding flow rates 1212. There may beany number of feeding time periods, as represented by ellipsis 1214.Following the desired number of feeding time periods 1214, process 1200may then terminate at END operation 1216. While FIG. 12 and process 1200show “increases” in the flow rates, other adjustments to the flow ratesmay be made. For example, the flow rates may decrease or remainsubstantially the same from one feeding period to the next.Considerations, such as metabolic activity, for example, may determinehow flow rates are adjusted. The “increases” in flow rates in FIG. 12are offered merely for illustrative purposes and are not intended to belimiting.

Turning to FIG. 13, example operational steps 1300 of a process forfeeding cells while retaining the cells in a first location, e.g., inthe bioreactor, that may be used with a cell expansion system, such asCES 500 (e.g., FIGS. 5B & 5C), are provided in accordance withembodiments of the present disclosure. START operation 1302 isinitiated, and process 1300 proceeds to load a disposable set onto thecell expansion system, prime the set, perform IC/EC washout, conditionmedia, and load cells, e.g., suspension or non-adherent cells, forexample. Next, process 1300 proceeds to feed the cells during a firsttime period 1304. In embodiments, a first inlet flow rate and a firstcirculation flow rate may be used. As an example, a first IC inlet flowrate and a first IC circulation flow rate may be used, in which thefirst IC inlet flow rate may be produced and controlled by the IC inletpump, e.g., first pump, and the first IC circulation flow rate may beproduced and controlled by the IC circulation pump, e.g., second pump.In an example embodiment, the first IC inlet pump rate may cause avolumetric flow rate of 0.1 mL/min to enter the IC inlet port of thebioreactor with a complementary IC circulation pump rate of −0.1 mL/mincausing a volumetric flow rate of −0.1 mL/min to enter the IC outletport of the bioreactor, in which the negative symbol (“−”) used in −0.1mL/min, for example, indicates a direction of the IC circulation pump(512) to cause or produce a counter-flow rate to maintain cells in thebioreactor during the growth phase of the cell culture. In anotherembodiment, the first IC circulation flow rate may be a percentage orfraction or portion of the first IC inlet flow rate. For example, thefirst IC circulation flow rate may be about fifty percent (50%) or aboutone-half (½), or another percentage or portion according to embodiments,of the IC inlet flow rate.

During the feeding (and expansion) of the cells and the use of the ICpumps to control cell residence in the bioreactor through flow andcounter-flow properties, the cells continue to grow and expand. As aresult, the cells may demand additional media, e.g., glucose and/or cellgrowth formulated media, to support the expanding population. Inembodiments, efforts may be made to increase the rate of media additionto feed the expanding cell population. In an example embodiment, anincrease in the IC inlet pump rate (+) may cause the IC inlet flow rateto increase by a first amount to achieve a second IC inlet flow rate1306. For example, the IC inlet flow rate may be increased by a firstamount of 0.1 mL/min to achieve a second IC inlet flow rate of 0.2mL/min, according to an embodiment. Other adjustments may be made to theIC inlet flow rate according to other embodiments.

Next, the second IC circulation flow rate may be set, or configured oradjusted, to equal a percentage or portion or fraction of the second ICinlet flow rate 1308, according to embodiments. For example, the secondIC circulation flow rate may be set, or configured or adjusted, to equalabout fifty percent (50%) or about one-half (½), or another percentageor portion according to embodiments, of the value of the IC inlet flowrate, according to an embodiment. In an embodiment, all, orsubstantially all, of the flow from the first fluid flow path 506 mayflow to IC inlet port (501A) of bioreactor 501 from connection 517, forexample. In another embodiment, all, or substantially all, of the flowfrom the first fluid flow path 506 may flow to IC outlet port (501B) ofbioreactor 501 from connection 517. In yet another embodiment, a firstportion of the flow from first fluid flow path 506 may flow fromconnection 517 to IC inlet port (501A), and a second portion of the flowfrom the first fluid flow path 506 may flow from connection 517 to ICoutlet port (501B). In embodiments, the percentage of the IC inlet flowrate at which the IC circulation flow rate may be set may range fromabout 0 percent to about 100 percent. In other embodiments, thepercentage may be between about 25 percent and about 75 percent. Inother embodiments, the percentage may be between about 40 percent andabout 60 percent. In other embodiments, the percentage may be betweenabout 45 percent and about 55 percent. In embodiments, the percentagemay be about 50 percent.

Depending on the value of the first IC circulation flow rate, anadjustment to the IC circulation pump rate (−) may cause the second ICcirculation flow rate to increase, according to an embodiment. Inanother embodiment, an adjustment may be made to the IC circulation pumprate to cause the second IC circulation flow rate to decrease such thatthe second IC circulation flow rate may be substantially equals to apre-defined percentage or pre-defined fraction of the second IC inletflow rate. In yet another embodiment, no adjustment may be made to theIC circulation pump rate. For example, where the first IC inlet flowrate equals 0.1 mL/min, and the first IC circulation flow rate equals−0.1 mL/min, if the second IC inlet flow rate is increased to 0.2mL/min, the second IC circulation flow rate may be set to (−½)*(secondQ_(IC Inlet)) or (−½)*(0.2 mL/min), which provides for a secondQ_(IC Circ) of −0.1 mL/min, and no adjustment to the IC circulation pumprate may be made to achieve such second Q_(IC Circ), according to anembodiment.

The cells may then be fed (and continue expanding) during a second timeperiod at the second IC inlet flow rate and at the second IC circulationflow rate 1310 to maintain the cells in the bioreactor and outside ofthe headers or portion of the IC circulation path outside of thebioreactor, for example. Following the second time period of feeding1310, process 1300 may terminate at END operation 1316 if it is notdesired to continue feeding and/or expanding the cells, for example.

In embodiments, a first time period, a second time period, a third timeperiod, a fourth time period, a fifth time period, etc. may eachcomprise one or more days (and/or hours and/or minutes). For example, atime period may be one (1) to fourteen (14) days, according toembodiments. However, a time period may be less than one (1) day orgreater than fourteen (14) days, in other embodiments. In an embodiment,a first time period for feeding may comprise Day 0, Day 1, Day 2, Day 3,Day 4; a second time period for feeding may comprise Day 5; a third timeperiod for feeding may comprise Day 6; and a fourth time period forfeeding may comprise Day 7, for example. In another embodiment, a firsttime period may comprise Day 0, Day 1, and Day 2 (e.g., duration ofabout 3 days); a second time period may comprise Day 3, Day 4, and Day 5(e.g., duration of about 3 days); a third time period may comprise Day6, Day 7, and Day 8 (e.g., duration of about 3 days); a fourth timeperiod may comprise Day 9 and Day 10 (e.g., duration of about 2 days);and a fifth time period may comprise Day 11, Day 12, and Day 13 (e.g.,duration of about 3 days). Time periods may be different durationsaccording to embodiments. Each time period may be measured in days,hours, minutes, and/or portions thereof.

Returning to FIG. 13, process 1300 may optionally continue to optionalquery 1312 to determine whether to adjust feeding rates or flow ratesbased on metabolic activity or metabolic levels. For the expansion ofTregs, for example, it may be desired to control the lactate values ofthe expanding cell population to values about 7 mmol/L. Where it isdesired to maintain the cell culture lactate values at or below about 7mmol/L, for example, the rate of media addition may be controlled duringthe expansion of the cells, e.g., regulatory T cells. In otherembodiments, it may be desired to maintain the lactate levels≤about 5mmol/L to improve cell growth and viability. In embodiments, graphicaluser interface (GUI) elements may be used to control the rate of mediaaddition and to maintain the lactate metabolic waste product fromglycolysis below a pre-defined level during the expansion of cells.

At optional query 1312, if it is not desired to measure metabolicactivity and/or adjust feeding levels based on such measurement(s),process 1300 proceeds “no” to END operation 1316, and process 1300terminates. Alternatively, where it is desired to adjust feeding levelsbased on metabolic activity, process 1300 proceeds “yes” to optionalstep 1314 to continue to increase the rate of media addition and feedthe expanding cell population. While step 1314 is shown as one step,this step may involve numerous adjustments to the media addition rate,such as increasing the IC inlet flow rate and increasing the ICcirculation flow rate, for example. Step 1314 is shown as one step onlyfor illustrative purposes and is not intended to be limiting. Followingany adjustments to the rate of media addition, process 1300 returns tooptional query 1312 to determine whether to continue to measuremetabolic levels and/or adjust feeding. If it is not desired to adjustfeeding levels based on metabolic activity, process 1300 proceeds “no”to END operation 1316, and process 1300 terminates. While FIG. 13 andprocess 1300 show “increases” in the rates or flow rates, otheradjustments to the flow rates may be made. For example, the rates orflow rates may decrease or remain substantially the same from onefeeding period to the next. The type of adjustments which may be mademay depend on the metabolic activity assessment of the growing cellpopulation. The “increases” in flow rate in FIG. 13 are offered merelyfor illustrative purposes and are not intended to be limiting.

Next, FIG. 14 illustrates example operational steps 1400 of a processfor retaining cells during feeding that may be used with a cellexpansion system, such as CES 500 (FIGS. 5B & 5C), in accordance withembodiments of the present disclosure. START operation 1402 isinitiated, and process 1400 proceeds to load a disposable tubing set orpremounted fluid conveyance assembly (e.g., 210 or 400) 1404 onto thecell expansion system. Next, the system may be primed 1406. In anembodiment, a user or operator, for example, may provide an instructionto the system to prime by selecting a task for priming, for example. Inan embodiment, such task for priming may be a pre-programmed task, forexample. Next, the IC/EC washout task may be performed 1408, in whichfluid on the IC circulation loop and on the EC circulation loop may bereplaced, for example. The replacement volume may be determined by thenumber of IC volumes and EC volumes exchanged, according to anembodiment.

Next, to maintain the proper or desired gas concentration across thefibers in the bioreactor membrane, the condition media task 1410 may beexecuted to allow the media to reach equilibrium with the provided gassupply before cells are loaded into the bioreactor. For example, contactbetween the media and the gas supply provided by the gas transfer module(GTM) or oxygenator may be provided by adjusting the EC circulationrate. The system may then be maintained in a proper or desired stateuntil a user or operator, for example, is ready to load cells into thebioreactor. In an embodiment, the system may be conditioned with media,such as complete media, for example. Complete media may be any mediasource used for cell growth. In an embodiment, the system may beconditioned with serum-free media, for example. In an embodiment, thesystem may be conditioned with base media. Any type of media understoodby those of skill in the art may be used.

Process 1400 next proceeds to loading cells 1412 into the bioreactorfrom a cell inlet bag, for example. In an embodiment, the cells in thecell inlet bag may be in solution with media to feed 1414 the cells, forexample. In another embodiment, the cells in the cell inlet bag may bein solution both with media to feed 1414 the cells and with a solubleactivator complex to stimulate the cells, e.g., T cells or Tregs. In anembodiment, the cells (and feed solution, in embodiments) may be loadedinto the bioreactor from the cell inlet bag until the bag is empty.Cells (and feed solution, in embodiments) may be chased from the airremoval chamber to the bioreactor. In an embodiment, a “load cells withuniform suspension” task may be executed to load the cells (and feedsolution, in embodiments). In another embodiment, a “load cellscentrally without circulation” task may be executed to load the cells(and feed solution, in embodiments) into a specific, e.g., central,region of the bioreactor. Other loading methods and/or loading tasks maybe used in accordance with embodiments.

Next, process 1400 proceeds to query 1416 to determine whether to use amodified feeding method to retain the cells, e.g., non-adherent orsuspension cells, such as T cells or Tregs, in the bioreactor, e.g.,hollow fiber bioreactor. For example, it may be desired to locate thecells in the bioreactor itself and out of the headers of the bioreactoror the rest of the IC loop. If it is not desired to use a modifiedfeeding method to retain the cells in the bioreactor itself, process1400 proceeds “no” to expand the cells 1426, in which the cells maycontinue to grow/expand using the media that they were initially fedwith in step 1414, for example.

On the other hand, if it is desired to retain the cells in thebioreactor itself, process 1400 proceeds “yes” to modified feed 1418, inwhich the cells may be fed by using a flow rate into the IC inlet port(501A) of the bioreactor (501) and a flow rate into the IC outlet port(501B) of the bioreactor (501) to keep the cells in the bioreactor. Inso doing, an inlet volumetric flow rate or inlet flow rate may beintroduced into the first fluid flow path (506) 1420. For example, an ICinlet flow rate may be introduced into the fluid flow path (506) 1420.The IC inlet pump (554), e.g., a first peristaltic pump (in anembodiment), may operate at a pre-defined number of revolutions perminute (RPMs) to cause a pre-defined IC inlet volumetric flow rate, orIC inlet flow rate, of fluid in the first fluid flow path (506) 1420. Aprocessor(s) and/or controller(s) may direct or control the first pumpand/or second pump, for example, to operate at a pre-defined number ofRPMs, according to an embodiment. Depending on the speed and directionof the IC circulation pump (512), a modified first flow rate, or firstportion of the IC inlet flow rate, may enter the IC inlet port (501A),or first port, of the bioreactor (501) 1422. A pump rate of a pump maydepend on the diameter of the pump or configuration of the pump, e.g.,peristaltic pump. Other types of pumps may also be used, in which thepump rate may depend on the configuration of the pump used. The ICcirculation pump (512), e.g., a second peristaltic pump (in anembodiment), may operate at a pre-defined number of RPMs, and in adirection opposite a direction of the first pump, to cause or produce apre-defined IC circulation flow rate, or second flow rate, or secondportion of the IC inlet flow rate, to enter the IC outlet port (501B),or second port, of the bioreactor (501) 1424. For example, embodimentsmay provide for about one-half (about ½), or a first portion, of the ICinlet flow rate to branch at connection 517 to enter IC inlet port(501A) of bioreactor 501, and about one-half (or ½), or a secondportion, of the IC inlet flow rate to branch at connection 517 to enterthe IC outlet port (501B) of bioreactor 501. As shown, the sum of thefirst portion and the second portion may substantially equal the ICinlet flow rate 503 (e.g., FIG. 5B), in which the IC inlet flow rate 503may be pumped by IC inlet pump 554. Depending on the flow andcounter-flow rates which may be used to keep the cells in the bioreactoror cell growth chamber 501, other types of fractions or percentages ofthe IC inlet pump rate may be used to set, or configure or adjust, theIC circulation pump, according to embodiments.

After feeding the cells with such flow and counter-flow properties tokeep the cells in the bioreactor, process 1400 proceeds to grow/expandthe cells 1426. While the expansion of cells is shown at step 1426, thecells may also grow/expand during one or more other step(s), such as1412, 1414, 1416, 1418, 1420, 1422, 1424, for example. From expand step1426, process 1400 proceeds to harvest or remove the cells 1430. Process1400 may then terminate at END operation 1432. If any other steps aredesired before harvest, such as continuing with a second modified feedmethod or other type of feeding method, process 1400 proceeds tooptional “Other” step 1428, according to embodiments. From optional step1428, process 1400 proceeds to harvest or remove the cells from thebioreactor 1430, and process 1400 may then terminate at END operation1432.

Turning to FIG. 15A, example operational steps 1500 of a process forfeeding cells to retain the cells in a first location, e.g., in thebioreactor itself, that may be used with a cell expansion system, suchas CES 500 (e.g., FIGS. 5B & 5C), are provided in accordance withembodiments of the present disclosure. START operation 1502 isinitiated, and process 1500 proceeds to load the disposable tubing setor premounted fluid conveyance assembly (e.g., 210 or 400) 1504 onto thecell expansion system. Next, the system may be primed 1506. In anembodiment, a user or operator, for example, may provide an instructionto the system to prime by selecting a task for priming, for example. Inan embodiment, such task for priming may be a pre-programmed task. Next,an IC/EC washout task may be performed 1508, in which fluid on the ICcirculation loop and on the EC circulation loop may be replaced, forexample. The replacement volume may be determined by the number of ICvolumes and EC volumes exchanged, according to an embodiment.

Next, to maintain the proper or desired gas concentration across thefibers in the bioreactor membrane, the condition media task 1510 may beexecuted to allow the media to reach equilibrium with the provided gassupply before cells are loaded into the bioreactor. For example, contactbetween the media and the gas supply provided by the gas transfer module(GTM) or oxygenator may be provided by adjusting the EC circulationrate. The system may then be maintained in a proper or desired stateuntil a user or operator, for example, is ready to load cells into thebioreactor. In an embodiment, the system may be conditioned with media,such as complete media, for example. Complete media may be any mediasource used for cell growth. In an embodiment, the system may beconditioned with serum-free media, for example. In an embodiment, thesystem may be conditioned with base media. Any type of media understoodby those of skill in the art may be used.

Process 1500 next proceeds to loading cells 1512 into the bioreactorfrom a cell inlet bag, for example. In an embodiment, the cells in thecell inlet bag may be in solution with media to feed the cells, forexample. In another embodiment, the cells in the cell inlet bag may bein solution both with media and with a soluble activator complex tostimulate the cells, e.g., T cells or Tregs. In an embodiment, the cellsmay be loaded into the bioreactor from the cell inlet bag until the bagis empty. Cells may be chased from the air removal chamber to thebioreactor. In an embodiment, a “load cells with uniform suspension”task may be executed to load the cells. In another embodiment, a “loadcells centrally without circulation” task may be executed to load thecells into a specific, e.g., central, region of the bioreactor. Otherloading methods and/or loading tasks may be used in accordance withembodiments.

Next, process 1500 proceeds to feed the cells per a first process duringa first time period 1514. In an embodiment, the cells may be fed at aminimum or low feed rate, for example, where the cell population isbeginning to grow/expand, and a minimum or low feed rate is able to meetthe feeding demands of such population. For example, an IC inlet pumprate of +0.1 mL/min to cause or produce a first fluid flow rate of 0.1mL/min may be used during such first time period. While this exampleprovides for a low or minimum feed rate of 0.1 mL/min, a low or minimumfeed rate may be greater than or equal to about 0.01 mL/min and lessthan or equal to about 0.1 mL/min, according to embodiments. Inembodiments, the low or minimum feed rate may be greater than 0.1mL/min. If it is desired to reduce the loss of cells from the hollowfiber membrane bioreactor during such first time period, the IC inletpump rate of +0.1 mL/min may be matched, or closely or substantiallymatched, to a complementary IC circulation pump rate of −0.1 mL/min tomaintain cells in the bioreactor during the growth phase of the cellculture. Other pump rates and resulting fluid flow rates may be used inother embodiments.

Next, process 1500 proceeds to query 1516 to determine whether to adjustthe feeding rate to retain the cells in the bioreactor itself while alsoaccounting for a growing cell population and increasing feeding demands,according to embodiments. For example, FIG. 15B shows an increasing feedrate in response to an increasing cell population. As shown in FIG. 15B,graph 1528 shows the cell number versus the IC flow rate for a run orprocedure on a cell expansion system, e.g., Quantum® Cell ExpansionSystem. In an embodiment, the IC flow rate comprises media for feedingthe cells, and may thus also be referred to as the IC media flow rate.The number of cells 1530 is shown versus the IC flow rate (mL/min) 1532.As shown, the IC flow rate increases from 0.1 mL/min to 0.2 mL/min to0.3 mL/min with an increase in the cells and the growing feeding demandsof an expanding cell population, according to an embodiment. In theembodiment shown, there may be a substantially linear relationship, asshown by line 1534, between the number of cells and the IC flow rate.

Returning to FIG. 15A and query 1516, if it is not desired to adjust thefeeding rate, process 1500 proceeds “no” to expand the cells 1520, inwhich the cells may continue to grow/expand using the media with whichthey were fed during the first time period 1514, for example. While theexpansion of cells is shown at step 1520, the cells may also grow/expandduring one or more other step(s), such as 1512, 1514, 1516, 1518, forexample. On the other hand, if it is desired to adjust the feeding ratewhile keeping the cells in the bioreactor, process 1500 proceeds “yes”to feed the cells per a second process during a second time period 1518.In an embodiment, such second process may involve feeding the cells atsubstantially the same feed rates as during the first time period, forexample. In another embodiment, the second process may involve feedingthe cells at different feed rates as compared to the feed rates usedduring the first time period. In an embodiment, the IC inlet flow ratemay be increased, and the IC circulation flow rate may be set, orconfigured or adjusted, to equal a percentage or portion or fraction ofthe IC inlet flow rate. For example, the IC circulation flow rate may beset to equal about fifty percent (50%) or about one-half (½), or anotherpercentage or portion according to embodiments, of the value of the ICinlet flow rate, according to an embodiment. Determining whether to set,configure or adjust, the IC circulation flow rate to a percentage orfraction or portion of the IC inlet flow rate may be based on the valueof the IC inlet flow rate, according to an embodiment. For example, anembodiment provides for the following method to retain cells in abioreactor when feeding the cells using IC inlet flow (whereQ_(IC Circ)=IC circulation flow rate (mL/min); Q_(IC Inlet)=IC Inletflow rate (mL/min)):

Q _(IC Circ)=(−)½*Q _(IC Inlet) when Q _(IC Inlet)≥0.2 mL/min

and

Q _(IC Circ)=(−)Q _(IC Inlet) when Q _(IC Inlet)=0.1 mL/min.

While the above equations provide for different calculations of the ICcirculation flow rate based on the values of the IC inlet flow rates(e.g., 0.2 mL/min or 0.1 mL/min), other values of such IC inlet flowrate for making such determination may be used, according to otherembodiments. Further, while about fifty percent (50%) or about one-half(½) is used in this example, other percentages, ratios, fractions,and/or portions may be used in accordance with embodiments. Returning toprocess 1500, after feeding the cells with such flow and counter-flowproperties as a part of second process 1518 to keep the cells in thebioreactor, process 1500 proceeds to grow/expand the cells 1520. Whilethe expansion of cells is shown at step 1520, the cells may alsogrow/expand during one or more other step(s), such as 1512, 1514, 1516,1518, for example. From expand step 1520, process 1500 proceeds toharvest or remove the cells 1524. Process 1500 may then terminate at ENDoperation 1526. If any other steps are desired before harvesting thecells, process 1500 proceeds to optional “Other” step 1522, according toembodiments. From optional step 1522, process 1500 proceeds to harvestor remove the cells from the bioreactor 1524, and process 1500 may thenterminate at END operation 1526.

Next, FIG. 16 illustrates example operational steps 1600 of a processfor feeding cells that may be used with a cell expansion system, such asCES 500 (e.g., FIGS. 5B & 5C), in accordance with embodiments of thepresent disclosure. START operation 1602 is initiated, in which adisposable set may be loaded onto a cell expansion system, the systemmay be primed, IC/EC washout may be performed, media may be conditioned,and cells may be loaded, for example. Process 1600 next proceeds to feedthe cells according to a first process during a first time period 1604.In an embodiment, the cells may be fed at a minimum or low feed rate,for example, where the cell population is beginning to grow/expand, anda minimum or low feed rate is able to meet the feeding demands of suchpopulation. For example, an IC inlet pump rate of +0.1 mL/min may beused during such first time period. While this example provides for alow or minimum feed rate of 0.1 mL/min, a low or minimum feed rate maybe greater than or equal to about 0.01 mL/min and less than or equal toabout 0.1 mL/min, according to embodiments. In embodiments, the low orminimum feed rate may be greater than 0.1 mL/min. If it is desired toreduce the loss of cells from the hollow fiber membrane bioreactorduring such first time period, the IC inlet pump rate of +0.1 mL/min maybe matched, or closely or substantially matched, to a complementary ICcirculation pump rate of −0.1 mL/min to maintain cells in the bioreactorduring the growth phase of the cell culture.

Next, process 1600 proceeds to query 1606 to determine whether thefeeding rate may be adjusted to account for a growing cell populationand/or to continue efforts to keep the cells in the bioreactor. If it isdesired to adjust the feeding rate to retain the cells in thebioreactor, process 1600 proceeds “yes” to feed the cells according to asecond process during a second time period 1608. In an embodiment, suchsecond process may involve feeding the cells at substantially the samefeed rates as during the first time period, for example. In anotherembodiment, the second process may involve feeding the cells atdifferent feed rates as compared to the feed rates used during the firsttime period. In an embodiment, the IC inlet flow rate may be increased,and the IC circulation flow rate may be set, or configured or adjusted,to equal a percentage or fraction or portion of the IC inlet flow rate.For example, the IC circulation flow rate may be set to equal aboutfifty percent (50%) or about one-half (½), or another percentage orportion according to embodiments, of the value of the IC inlet flowrate, according to an embodiment. Determining whether to set the ICcirculation flow rate to a percentage of the IC inlet flow rate may bebased on the value of the IC inlet flow rate, according to anembodiment. For example, an embodiment provides for the following (whereQ_(IC Circ)=IC circulation flow rate (mL/min), Q_(IC Inlet)=IC Inletflow rate (mL/min)):

Q _(IC Circ)=(−)½*Q _(IC Inlet) when Q _(IC Inlet)≥0.2 mL/min

and

Q _(IC Circ)=(−)Q _(IC Inlet) when Q _(IC Inlet)=0.1 mL/min.

While the above equations provide for different calculations of the ICcirculation flow rate based on the values of the IC inlet flow rates(e.g., 0.2 mL/min or 0.1 mL/min), other values of such IC inlet flowrate for making such determination may be used, according to otherembodiments. Further, while about fifty percent (50%) or about one-half(½) is used in this example, other percentages, ratios, fractions,and/or portions may be used in accordance with embodiments. Returning toprocess 1600, after feeding the cells with such flow and counter-flowproperties as a part of second process 1608 to keep the cells in thebioreactor, process 1600 proceeds to query 1610 to determine whether tomonitor or measure the metabolic activity, e.g., glucose consumptionand/or lactate generation, of the growing cell population. If it is notdesired to monitor the metabolic activity, process 1600 proceeds “no” toexpand the cells 1616, in which the cells may continue to grow/expandusing the media with which they were fed during the first time period1604 and/or second time period 1608, for example. While the expansion ofcells is shown at step 1616, the cells may also grow/expand during oneor more other step(s), such as 1604, 1606, 1608, 1610, 1612, 1614, forexample.

Returning to query 1610, if it is desired to monitor or measure themetabolic activity of the growing cell population, process 1600 proceeds“yes” to either continue to feed the cells per the second process oradjust the feed rate, based on the metabolic activity and/ormeasurements thereof. In an embodiment, monitoring the glucose and/orlactate levels may facilitate the adjustment of media flow rates, e.g.,IC flow rate, to support cell, e.g., T cell or Treg, expansion in abioreactor, e.g., hollow fiber bioreactor. In embodiments, cell culturelactate values may be maintained below about 7 mmol/L, for example. Inembodiments, by using a cell expansion system graphical user interface(GUI), for example, to control a rate(s) of media addition, lactatemetabolic waste product from glycolysis may be maintained below about 7mmol/L, for example, during the expansion of cells, e.g., regulatory Tcells. In other embodiments, rate(s) of media addition, for example,and/or other settings may be controlled to attempt to maintain thelactate levels≤about 5 mmol/L, for example, to improve cell growth andviability. Other concentrations may be used in other embodiments.

Depending on the metabolic measurements and the desired levels oflactate, for example, process 1600 proceeds to either continue feedingthe cells according to the second process 1612 or adjusting the feedrate 1614. For example, the cells may be continued to be fed accordingto the second process 1612 where measurements of the metabolic activityshow lactate levels≤about 5 mmol/L, according to an embodiment. Inanother embodiment, the cells may be continued to be fed according tothe second process 1612 where the measurements of the metabolic activityshow lactate levels≤about 7 mmol/L, for example. From continuing to feedthe cells per the second process 1612, process 1600 returns to query1610 to continue monitoring the metabolic activity of the growing cellpopulation.

Depending on the metabolic measurements and the desired levels thereof,process 1600 proceeds to adjust the feed rate 1614, in which the cellsmay be fed according to an additional process during an additional timeperiod. Such additional process(es) and additional time period(s) mayinclude, for example, a third process during a third time period, afourth process during a fourth time period, a fifth process during afifth time period, etc., according to embodiments. In an embodiment,such additional process may involve feeding the cells at substantiallythe same feed rates as during the first and/or second time periods, forexample. In another embodiment, the additional process may involvefeeding the cells at different feed rates as compared to the feed ratesused during the first and/or second time periods. For example, the ICinlet flow rate may be increased, and the IC circulation flow rate maybe matched, or closely or substantially matched, to the IC inlet flowrate, but in the opposite direction, in an embodiment. In anotherembodiment, the IC inlet flow rate may be increased, and the ICcirculation flow rate may be set, or configured or adjusted, to equal apercentage or fraction or portion of the IC inlet flow rate, and in theopposite direction. While adjusting the feed rate 1614 shows an“additional” process and an “additional” time period in step 1614, anynumber of processes and time periods may be used to adjust the feed ratebased on metabolic activity.

From adjusting the feed rate 1614, process 1600 returns to query 1610.If it is not desired to adjust, or further adjust, the feeding rate,process 1600 proceeds “no” to expand the cells 1616, in which the cellsmay continue to grow/expand using the media with which they were fedduring the first time period 1604, second time period 1608, and/oradditional time period 1614. While the expansion of cells is shown atstep 1616, the cells may also grow/expand during one or more otherstep(s), such as step(s) 1604, 1606, 1608, 1610, 1612, 1614, forexample. From expand step 1616, process 1600 proceeds to harvest orremove the cells 1618 from the bioreactor and into a harvest bag(s) orcontainer(s), for example. Process 1600 may then terminate at ENDoperation 1622. Alternatively, from harvest operation 1618, process 1600may optionally proceed to allow for further processing/analysis 1620.Such optional further processing/analysis 1620 may includecharacterizing the phenotype(s), for example, of the harvested cells,e.g., T cells or Tregs. From optional further processing/analysis step1620, process 1600 may then terminate at END operation 1622.

FIG. 17A illustrates operational steps 1700 of a process for expandingcells that may be used with a cell expansion system in embodiments ofthe present disclosure. As described below, process 1700 may includesteps to shear cells that have been expanded in the cell growth chamberaccording to embodiments of the present disclosure. In embodiments,these steps may be implemented as part of a “modified circulation” task.START operation 1702 is initiated and process 1700 proceeds to loadingfluid 1704 with cells into a cell growth chamber in a cell expansionsystem. In embodiments, the cells may comprise non-adherent cells, suchas one or more types of T cells. In one embodiment, the cells includeTregs.

Process 1700 proceeds to exposing the cells to an activator 1706. Theactivator, which may include antibody complexes, may be added to thefluid loaded at step 1704. In embodiments, the activator may be asoluble human antibody CD3/CD28/CD2 cell activator complex. Process 1700proceeds to expanding 1708 the cells for a first time period. Step 1708may include feeding 1710 the cells. The cells may be fed nutrients topromote their expansion. For example, media with glucose, proteins, andreagents may be delivered into the cell growth chamber to providenutrients for cell expansion.

The first time period for expanding 1708 the cells may be based on thetime it may take for cell colonies, micro-colonies, or clusters to form.A cell colony, micro-colony, or cluster may be a group of one or moreattached cells. In embodiments, the cells, e.g., Tregs, may benefit fromcell contact. The cell contact may stimulate signaling that promotesexpansion and growth. However, after a period of expansion, the cellsmay attach to each other to form cell colonies, micro-colonies, orclusters. Without being bound by theory, it is believed that after atime period of the cells expanding 1708 the cells may form relativelylarge cell colonies, micro-colonies, or clusters that continue to grow.The cell colonies, micro-colonies, or clusters may create necroticcenters where nutrients (e.g., glucose), gasses (e.g., oxygen), andreagents (e.g., activator) do not reach cells in the center of the cellcolonies, micro-colonies, or clusters. As a result, the conditions forcell expansion in the center of these cell colonies, micro-colonies, orclusters may be such that the expansion rate may slow (e.g., increasedoubling time) or the conditions may lead to cell necrosis.

In embodiments, to allow the expansion 1708 of the cells, the first timeperiod may be between about 5 hours and about 48 hours. In someembodiments, the first time period may be greater than about 6 hours,greater than about 12 hours, greater than about 24 hours, or evengreater than about 48 hours. In other embodiments, the first time periodmay be less than about 72 hours, less than about 60 hours, less thanabout 48 hours, less than about 36 hours, less than about 24 hours, oreven less than about 12 hours. After the first time period, process 1700proceeds to circulate 1712 to shear cell colonies, micro-colonies, orclusters during a second time period. Step 1712 may be performed toreduce the size of the cell colonies, micro-colonies, or clusters. Thesecond time period may in embodiments be less than about 120 minutessuch as between about 60 minutes and about 0.5 minutes. In otherembodiments, the second time period may be based on a volume of fluidintroduced into the first circulation path.

FIG. 17B illustrates a number of views 1750, 1760, and 1770 of cells ina volume of fluid (1752) that may be expanded in a cell growth chamberas part of process 1700. For example, in some embodiments, the cellgrowth chamber may be a hollow fiber bioreactor. In these embodiments,views 1750, 1760, and 1770 may illustrate cells in a fiber of a hollowfiber bioreactor, for example. 1750A, 1760A, and 1770A are zoomed inportions of views 1750, 1760, and 1770, respectively. Referring to view1750, the cells may be shown after cells have been loaded 1704, exposedto an activator, 1706 and expanded 1710 for a time period, e.g., thefirst time period. As illustrated in view 1750, a number of cellcolonies 1754A-E have formed.

In order to reduce the number of cells in, and the size of, the cellcolonies, micro-colonies, or clusters 1754A-E, step 1712 may circulatefluid and the cells through a first fluid circulation path. Withoutbeing bound by theory, it is believed that the circulation may createsome force acting on the cell colonies including shear stress, asillustrated by arrow 1756 as shown in zoomed in portion 1760A. The shearstress 1756 may provide enough force to separate cells in the cellcolonies. As the circulation continues, the cell colonies may begin tobreak up into smaller sizes, as shown in view 1760. View 1770illustrates the cells after the circulation has been performed for thesecond time period. As illustrated in view 1770, the size of the cellcolonies are reduced with some colonies being completely separated intoindividual cells. In some embodiments, the circulation to shear step1712 may be performed until the cells and fluid comprise a single cellsuspension.

In other embodiments, cell colonies, micro-colonies, or clusters ofcells may remain after circulate to shear 1712. For example, colony1754F in zoomed in portion 1770A illustrates that some colonies of areduced size may remain after step 1712. In embodiments the cellcolonies, micro-colonies, or clusters (e.g., 1754F) that remain may bebetween about 25 microns and about 300 microns. In other embodiments,circulate to shear 1812 may reduce the size of cell colonies,micro-colonies, or clusters (e.g., 1754F) so the cell colonies,micro-colonies, or clusters may be between about 50 microns and about250 microns. In yet other embodiments, step 1712 may reduce the size ofcell colonies, micro-colonies, or clusters to between about 75 micronsand about 200 microns. In some embodiments, the size of the cellcolonies, micro-colonies, or clusters may be less than about 200 micronse.g., about 100 microns after step 1712.

In embodiments, the size of the remaining cell colonies, micro-colonies,or clusters may be somewhat a function of some structural features ofthe cell growth chamber. As mentioned above, the cell growth chamber maybe a hollow fiber bioreactor with hollow fibers in some embodiments. Asmay be appreciated, cell colonies, micro-colonies, or clusters, as theycirculate, may be affected by shear stress each time they contact theside walls of the hollow fiber. This contact may more efficiently reducethe size of cell colonies. When the inner diameter is larger, such as ina conventional process that may utilize a pipet to induce shear stressto reduce colony sizes, contact with a side wall may not occur as often.FIG. 17C illustrates differences in inner diameter size between oneembodiment of a hollow fiber (e.g., 215 microns) 1772 and a pipet tip1774 (762 microns), which may be used to disassociate attached cells incell colonies, micro-colonies, or clusters. In embodiments, the smallerinner diameter of a hollow fiber is believed to more efficiently andeffectively reduce a size of cell colonies during the circulating toshear step 1712.

The second time period for the circulate to shear 1712 may inembodiments be less than about 120 minutes, less than about 90 minutes,less than about 60 minutes, less than about 30 minutes, or even lessthan about 15 minutes. In some embodiments, the second time period maybe between about 15 minutes and about 1 minute, such as about 4 minutes.

After the second time period, process 1700 proceeds to move cells intocell growth chamber during a third time period 1714. At step 1714 cellsthat are not positioned in the cell growth chamber, as a result of thecirculation to shear step 1712, are moved back into the cell growthchamber during a third time period. In embodiments, this may involveactivating one or more pumps to introduce fluid into a fluid circulationpath. For example, fluid may be introduced from a fluid inlet path to afirst fluid flow path and then into the cell growth chamber, from bothan inlet port and an outlet port of the cell growth chamber. Themovement of the fluid into the cell growth chamber from the inlet portand the outlet port may move cells back into the cell growth chamber.

In some embodiments, the fluid used in the step to move the cells backinto the cell growth chamber 1714 may include reagents that promote cellgrowth. For example, in embodiments, the fluid may be media thatincludes glucose, proteins, or other reagents. In one embodiment, thefluid may include one or more supplements. In one embodiment, the fluidis complete media and includes a cytokine, e.g., human IL-2 cytokinesupplement. The addition of the fluid may be referred to as a bolusaddition. The combination of steps 1712 and 1714 may be referred to inembodiments as circulate and bolus addition.

In other embodiments, the third time period may be based on a volume offluid introduced into the cell growth chamber during the circulate toshear step 1712. For example, in embodiments, step 1712 may be performeduntil about 300 ml, about 250 ml, about 200 ml, or about 150 ml, havebeen introduced into the fluid circulation path.

After the third time period, process 1700 proceeds to expand during afourth time period 1716. Similar to step 1708, step 1716 may includefeeding 1718 the cells. The cells may be fed nutrients to promote theirexpansion. For example, media with glucose, proteins, and reagents maybe delivered into the cell growth chamber to provide nutrients for cellexpansion.

Similar to the first time period, the fourth time period may be based onthe time it may take for cell colonies to form. In embodiments, thefourth time period may be between about 5 hours and about 48 hours. Insome embodiments, the fourth time period may be greater than about 6hours, greater than about 12 hours, greater than about 24 hours, or evengreater than about 48 hours. In other embodiments, the fourth timeperiod may be less than about 72 hours, less than about 60 hours, lessthan about 48 hours, less than about 36 hours, less than about 24 hours,or even less than about 12 hours. Because more cells are likely in thecell growth chamber, the fourth time period may be shorter than thefirst time period in some embodiments.

After the fourth time period, process 1700 proceeds to step 1720 tocirculate to shear for a fifth time period to reduce second cellcolonies. In embodiments, step 1720 may use the first circulation rate.However, in other embodiments, the circulation rate used at step 1720may be different, either greater or less than the first circulationrate.

After the fifth time period, process 1700 proceeds to step 1722 wherethe cells that are not positioned in the cell growth chamber, may bemoved back into the cell growth chamber during a sixth time period.Fluid may be introduced from a fluid inlet path to a first fluid flowpath and into the cell growth chamber from an inlet port and an outletport of the cell growth chamber. The movement of the fluid into the cellgrowth chamber from the inlet port and the outlet port may move cellsback into the cell growth chamber. In some embodiments, the fluid usedto move the cells back into the cell growth chamber may include reagentsthat promote cell growth. For example, in embodiments, the fluid may bemedia that includes glucose, proteins, or other reagents. In oneembodiment, the fluid may include one or more supplements. In oneembodiment, the fluid is complete media and includes a cytokine, e.g.,human IL-2 cytokine.

The process 1700 may optionally perform the steps of expand, circulate,and move cells for an additional number of times as illustrated byoptional step 1724 and ellipsis 1726. The steps of expand, circulate,and move may be performed sequentially for a period of time. Forexample, in some embodiments, the steps may be performed once every fourdays, once every three days, once every two days, daily, twice daily, orthree times daily, for a period of from about two days to about twentydays (such as about 10 days). In some embodiments, the steps may beperformed at varying periods of time. For example, in one embodiment,the steps may be performed after three days, and then every other day.As another example, the steps may be performed after two days and thentwice daily. These are merely examples and other embodiments may utilizeother periods of time.

For example, FIG. 18 shows a graph 1800 of performing circulation andbolus additions at different periods of time during a process ofexpanding cells. Curve 1808 shows the cell number 1802 versus days ofcell culture 1804 on a cell expansion system, e.g., Quantum® CellExpansion System. Curve 1806 shows IC Flow rate 1818 versus days of cellculture 1804 on a cell expansion system, e.g., Quantum® Cell ExpansionSystem. As shown by curve 1806, the IC flow rate remains the same at 0.1mL/min for the first three days. There is an increase in the flow rateto 0.2 mL/min at day six and an increase to 0.3 mL/min after day seven.The increase in flow rate may be in response to the increase in cellnumbers as the culture days 1804 increase. Also, shown in FIG. 18, areseveral circulation and bolus addition steps (e.g., 1810, 1812, 1814,and 1816). There is a circulation and bolus addition 1810 performed 3.5days after cell culture. There is another circulation and bolus addition1812 performed after 4.5 days of cell culture. Another circulation andbolus addition is performed after 6 days of cell culture 1814, andanother circulation and bolus addition after 6.5 days of cell culture1816. As may be appreciated by looking at curves 1806 and 1808, themultiple circulation and bolus additions (1810-1816), in combinationwith the increasing IC flow rates, may have a positive effect on therate of cell expansion, e.g., number of cells.

Referring back to 17A, process 1700 proceeds to remove cells 1728 fromthe cell growth chamber. In embodiments, this may involve harvesting thecells. Step 1728 may include additional steps such as circulation steps(e.g., 1712 and 1720) prior, or during, removal of the cells from thecell growth chamber. Process 1700 terminates at END operation 1730.

FIG. 19A illustrates operational steps 1900 of a process for operatingpumps that may be used in a cell expansion system in embodiments of thepresent disclosure. As described below, process 1900 may include stepsto activate pumps in a process of reducing cells in cell clusters thathave been expanded in the cell growth chamber according to embodimentsof the present disclosure. In embodiments, these steps may beimplemented as part of a “modified circulation” task. In embodiments,the steps of process 1900 may be performed by a computer processor.START operation 1902 is initiated and process 1900 proceeds to step1904, where a first pump is activated at a first flow rate to introducea first volume of fluid including cells into the intracapillary portionof a bioreactor of a cell expansion system. In embodiments, the firstpump may be an inlet pump.

After activating the first pump at the first flow rate, process 1900proceeds to the first pump activated at a second flow rate 1906 tointroduce media with nutrients into the intracapillary portion of thebioreactor for a first time period. The nutrients may include forexample proteins, glucose, and other compounds that are used to feed andpromote the expansion of the cells. For example, referring to FIG. 19B,a first pump 1960 may be activated at the second flow rate to introducemedia into an inlet port 1962A of bioreactor 1962.

In embodiments, the first time period may be based on the time it maytake for cell colonies to form. In embodiments, the first time periodmay be between about 5 hours and about 48 hours. In some embodiments,the first time period may be greater than about 6 hours, greater thanabout 12 hours, greater than about 24 hours, or even greater than about48 hours. In other embodiments, the first time period may be less thanabout 72 hours, less than about 60 hours, less than about 48 hours, lessthan about 36 hours, less than about 24 hours, or even less than about12 hours.

Process 1900 then proceeds to activating a second pump at a third flowrate to direct fluid into the bioreactor 1908. Optional step 1908 may beperformed to activate pump 1964 to direct a portion of the fluid,introduced at step 1906, into an outlet port 1962B of the bioreactor1962 to feed cells, for example.

Process 1900 then proceeds to activating the second pump at a fourthflow rate 1910 to circulate the cells during a second time period andreduce a number of cells in a cell cluster in the bioreactor. Inembodiments, the cells are circulated throughout a first fluidcirculation path. Referring to FIG. 19C, second pump 1964 may beactivated at step 1910 to circulate fluid through first fluidcirculation path 1966, s illustrated by arrows 1968A-D.

Without being bound by theory, it is believed that after a time periodthe expanding cells may form cell colonies, micro-colonies, or clusters.The cell colonies may create necrotic centers where nutrients andproteins (e.g., activator) do not reach cells in the center of thecolonies. As a result, the conditions for cell expansion in the centerof these cell colonies, micro-colonies, or clusters may be such that theexpansion rate may slow (e.g., increase doubling time) and may result incell necrosis. Step 1910 may be performed to reduce the size of the cellcolonies, micro-colonies, or clusters.

In embodiments, the fourth flow rate may be high enough to induceshearing. For example, in embodiments, the fourth flow rate may be ashigh as about 1000 ml/min. In other embodiments, the fourth flow ratemay be between about 100 mL/min and about 600 ml/min, such as forexample, 300 mL/min.

After the second time period, process 1900 proceeds to step 1912, wherethe first pump is activated at a fifth flow rate to introduce fluidthrough a first fluid flow path for a third time period. A first portionof the fluid introduced in the first fluid flow path may in embodimentsmove first cells in the first fluid flow path (that may be in the firstfluid flow path because of step 1910) back into the cell growth chamberthrough the inlet port 1962A. Referring to FIG. 19D, pump 1960 may beactivated to introduce fluid into the first fluid flow path 1970. Asillustrated by arrow 1968A and 1968B, fluid flows from the first fluidflow path 1970 into inlet port 1962A. This moves first cells that areoutside bioreactor 1962 back into bioreactor 1962. It is believed thatmoving cells that are in the first fluid flow path back into the cellgrowth chamber improves the overall expansion of cells, since theconditions for cell growth are optimized in the cell growth chamber.

In some embodiments, the fluid introduced into the first fluid flow pathat step 1912, may include one or more materials (e.g., reagents) thatpromote cell expansion. For example, in embodiments, the fluid may bemedia that includes glucose or other nutrients for feeding the cells. Inone embodiment, the fluid may include a reagent, which may compriseadditional activator for continuing to activate the expansion of cells.The use of the fluid with particular reagent(s) or other materials tomove the cells back into the cell growth chamber, and also expose thecells to additional reagents, e.g., growth factors, proteins, etc., thatpromote expansion may provide improved cell expansion. In embodiments,the additional fluid used in step 1912 may be referred to as a bolusaddition.

Also, after the third time period, at step 1914, the second pump may beactivated at a sixth flow rate to move a second portion of the fluidintroduced through the first fluid flow path, and second cells, into thecell growth chamber through the outlet port 1962B. The second portion ofthe fluid may in embodiments move second cells in the first fluid flowpath (that may be in the first fluid flow path because of step 1910)back into the cell growth chamber through the outlet port. Referring toFIG. 19D, pump 1964 may be activated to move fluid introduced into thefirst fluid flow path 1970 to the outlet port 1962B. As illustrated byarrow 1968C, pump 1964 moves fluid and cells in first fluid circulationpath into bioreactor 1962 through outlet port 1962B. As illustrated, byFIG. 19D, the sixth flow rate may be in a direction opposite the fourthflow rate (FIG. 19C). This fluid movement moves cells that are outsidebioreactor 1962 back into bioreactor 1962.

In embodiments, the sixth flow rate may be less than the fifth flowrate, which as noted above moves fluid into the first fluid flow path.As may be appreciated, the fifth flow rate may result in introduction ofa volume of fluid into a portion of the circulation path 1966 based onthe fifth flow rate. The sixth rate may be set so that a percentage ofthat volume moves toward the outlet port 1962B.

In embodiments, the sixth flow rate may be set as a percentage of thefifth flow rate. For example, the sixth flow rate may be less than orequal to about 90% of the fifth flow rate. In some embodiments, thesixth flow rate may be set to less than or equal to about 80% of thefifth flow rate. In other embodiments, the sixth flow rate may be lessthan or equal to about 70% of the fifth flow rate. In yet otherembodiments, the sixth flow rate may be less than or equal to about 60%of the fifth flow rate. In some embodiments, the sixth flow rate may beless than or equal to about 50% of the fifth flow rate.

In embodiments, the sixth flow rate is based, at least in part, on thedifference between a first volume, between the second pump and the inletport, and a second volume, between the second pump and the outlet port.Referring to FIG. 19D, portions of the first circulation path may havedifferent volumes. For example, in one embodiment, a first volumebetween the second pump 1964 and the inlet port 1962A may have a firstvolume, and a second volume between the second pump 1964 and the outletport 1962B may have a second volume that is different from the first,e.g., larger. In these embodiments, in order to move the cells from thecirculation path back into the bioreactor, so that the cells generallyreach the bioreactor at approximately the same time, the sixth flow ratemay be set at least in part based on the differences in these volumes.

As may be appreciated, as fluid enters fluid circulation path 1966 fromfirst fluid flow path 1970, the fluid moves toward inlet port 1962A atthe flow rate that first pump 1960 is set. When pump 1964 is activated,it will redirect at least a portion of the fluid toward the outlet port1962B. In embodiments, the second volume (the volume between 1964 andoutlet port 1962B) may be larger than the first volume. Therefore, inorder to move more fluid into the second volume, the second pump 1964may be set at a percentage of the rate of pump 1960.

In one embodiment, at step 1912, pump 1960 may be set to 100 ml/min. Inthis embodiment, the second volume (from pump 1964 to outlet port 1962B)may be larger than the first volume (from pump 1964 to inlet port1962A). In order to account for the additional volume, embodiments mayprovide for pump 1964 to be set at 70 ml/min during step 1914. Thisembodiment may provide for cells to reach bioreactor 1962 during thethird time period at about the same time.

In embodiments, process 1900 may optionally perform the steps of1906-1914 a number of additional times as illustrated by ellipsis 1916and optional step 1918. The steps of Activate: First Pump (at SecondRate) 1906, Second Pump (at Fourth Rate) 1910, First Pump (at FifthRate) 1912, and Second Pump (at Sixth Rate) 1914 may be continuouslyperformed for a period of time. As noted above, the steps may beperformed to feed cells, circulate cells to break up cell colonies,micro-colonies, or clusters, and move cells back into a cell growthchamber. For example, in some embodiments, the steps may be performedevery three days, every two days, daily, twice daily, or three times aday, for a period of from about two days to about twenty days (such as10 days). In some embodiments, the steps may be performed at varyingperiods of time. For example, in one embodiment, the steps may beperformed after three days, and then every other day. As anotherexample, the steps may be performed after two days and then twice daily.This is merely one example and other embodiments may utilize otherperiods of time. Process 1900 terminates at END operation 1920.

It is noted that in some embodiments, process 1900 may includeadditional steps. For example, a rocking device may be connected to thebioreactor and after the first time period (and during the second timeperiod), when the first pump is activated at step 1910, the rockingdevice may be activated to rotate the bioreactor as part of circulatingthe cells to reduce a number of cells in a cell cluster. This is merelyone example and other embodiments of process 1900 are not limitedthereto.

FIG. 20 illustrates example operational steps 2000 of a process forexpanding cells that may be used with a cell expansion system, such asCES 500 (e.g., FIG. 5A) or CES 600 (FIG. 6), in accordance withembodiments of the present disclosure. START operation 2002 isinitiated, and process 2000 proceeds to load the disposable tubing set2004 onto the cell expansion system. Next, the system may be primed2006. In an embodiment, a user or an operator, for example, may providean instruction to the system to prime by selecting a task for priming,for example. In an embodiment, such task for priming may be apre-programmed task. Process 2000 then proceeds to the IC/EC Washouttask 2008, in which fluid on the IC circulation loop and on the ECcirculation loop is replaced. The replacement volume is determined bythe number of IC Volumes and EC Volumes exchanged.

Next, to maintain the proper or desired gas concentration across thefibers in the bioreactor membrane, the condition media task 2010 may beexecuted to allow the media to reach equilibrium with the provided gassupply before cells are loaded into the bioreactor. For example, rapidcontact between the media and the gas supply provided by the gastransfer module or oxygenator is provided by using a high EC circulationrate. The system may then be maintained in a proper or desired stateuntil a user or operator, for example, is ready to load cells into thebioreactor. In an embodiment, the system may be conditioned withcomplete media, for example. Complete media may be any media source usedfor cell growth. In an embodiment, complete media may comprise alpha-MEM(α-MEM) and fetal bovine serum (FBS), for example. Any type of mediaunderstood by those of skill in the art may be used.

Process 2000 next proceeds to loading cells centrally withoutcirculation 2012 into the bioreactor from a cell inlet bag, for example.In embodiments, a “load cells centrally without circulation” task may beused, in which a first volume of fluid at a first flow rate comprising aplurality of cells may be loaded into the cell expansion system, inwhich the cell expansion system comprises a cell growth chamber. Asecond volume of fluid at a second flow rate comprising media may thenbe loaded into a portion of a first fluid circulation path, for example,to position the first volume of fluid in a first portion of the cellgrowth chamber. In an embodiment, the first portion of the cell growthchamber or bioreactor may comprise about a central region of thebioreactor. In an embodiment, the first volume is the same as the secondvolume. In an embodiment, the first flow rate is the same as the secondflow rate. In another embodiment, the first volume is different from thesecond volume. In another embodiment, the first flow rate is differentfrom the second flow rate. In an embodiment, the sum of the first volumeand the second volume may equal a percentage or proportion of thevolume, e.g., total volume, of the first fluid circulation path, forexample. For example, the sum of the first volume and the second volumemay be about 50% of the volume, e.g., total volume, of the first fluidcirculation path, for example. In an embodiment, fluid in the firstfluid circulation path flows through an intracapillary (IC) space of abioreactor or cell growth chamber. In an embodiment, fluid in a secondfluid circulation path flows through an extracapillary (EC) space, forexample, of a cell growth chamber or bioreactor. In an embodiment, thesum of the first volume and the second volume may be about 50%, oranother percentage or proportion according to embodiments, of the volumeof the intracapillary (IC) loop, for example. In an embodiment, the sumof the first volume and the second volume may be about 50%, or anotherpercentage or proportion according to embodiments, of the volume ofanother fluid path, loop, etc., as applicable. Other percentages orproportions may be used, including, for example, any percentage betweenand including about 1% and about 100%, in accordance with embodiments.

Following the loading of the cells 2012, process 2000 next proceeds tofeed the cells 2014. The cells may be grown/expanded 2016. While step2016 is shown after step 2014, step 2016 may occur before, orsimultaneous with, step 2014, according to embodiments. Next, process2000 proceeds to query 2018 to determine whether any cell colonies,micro-colonies, or clusters have formed. A cell colony, micro-colony, orcluster may be a group of one or more attached cells. If a cell colony,micro-colony, or cluster has formed, process 2000 proceeds “yes” toshear 2020 any cell colonies, micro-colonies, or clusters. For example,after expanding a plurality of cells for a first time period, the cellsmay be circulated at a first circulation rate during a second timeperiod to reduce a number of cells in a cell colony, micro-colony, orcluster. In embodiments, the circulating the cells at the firstcirculation rate may cause the cell colony to incur a shear stress, inwhich one or more cells in the cell colony may break apart from the cellcolony. In an embodiment, reducing the number of cells in the cellcolony, micro-colony, or cluster may provide a single cell suspension,for example. In embodiments, circulating the cells to shear any colony,micro-colony, or cluster 2020 may be used every two (2) days, forexample, during cell culture to maintain uniform cell density andnutrient diffusion. Other time periods may also be used according toembodiments. In an embodiment, such shearing of any micro-colonies,colonies, or clusters may begin on or after Day 4, for example. Otherdays or time periods on which to begin such shearing may be usedaccording to embodiments. Following shearing 2020, process 2000 may nextreturn to feed cells 2014.

If it is determined at query 2018 not to shear any cell colonies orclusters, or if none exist, for example, process 2000 proceeds “no” toresuspend cells 2022. In embodiments, circulating the cells may be usedto uniformly resuspend those cells that may be loosely adhered duringculture. In embodiments, step 2022 may include circulating the cells touniformly resuspend those cells that may be loosely adhered prior toinitiating a harvest task, or other task to remove cells from thebioreactor. Following the resuspension of the cells 2022, process 2000next proceeds to harvest the cells 2024. Further processing of theremoved cells or other analysis may optionally be performed at step2026, and process 2000 may then terminate at END operation 2028. If itis not desired to perform further processing/analysis, process 2000terminates at END operation 2030.

Turning to FIG. 21 and process 2100, START operation is initiated 2102,and process 2100 proceeds to load a disposable set 1204 onto a cellexpansion system, according to embodiments. The disposable set may thenbe primed 2106, and an IC/EC washout step 2108 may occur. The media maynext be conditioned 2110. Next process 2100 proceeds to load cells 2112,e.g., suspension or non-adherent cells, such as T cells or Tregs. In anembodiment, such cells may be loaded 2112 by a “load cells centrallywithout circulation” task. In another embodiment, such cells may beloaded 2112 by a “load cells with uniform suspension” task.

Next, process 2100 proceeds with beginning to feed the cells 2114, whichmay begin on Day 0, according to embodiments. The cells may grow andexpand 2116, and by Day 3, for example, it may be desired to add a bolusof a fluid to the IC loop and re-distribute the cells 2118. In anembodiment, such bolus of fluid may comprise a reagent, such ascytokines or other growth factor(s). In another embodiment, such bolusof fluid may comprise a reagent and base media, for example.

Following such bolus addition and re-distribution of cells, process 2100next proceeds to feeding 2120 the cells again, in which such feeding mayoccur on Day 3, for example. With such feeding 2120, the parameters ofthe system, such as one or more pumps controlling flow rate, may becontrolled 2122 to achieve complementary flow and counter-flow settingsfor fluid moving into the bioreactor from both the IC inlet port and theIC outlet port of the bioreactor. For example, the IC inlet pump 2124may be adjusted or directed to produce a flow, and the IC circulationpump may be adjusted or directed 2126 to produce a counter-flow. Forexample, an IC inlet pump rate of 0.1 mL/min may be matched, or closelyor substantially matched, to a complementary IC circulation pump rate of−0.1 mL/min to maintain cells in the bioreactor during the growth phaseof the cell culture, which may be Days 4-7, for example, in embodiments.Such control of settings 2122 may allow for counteracting any forcesassociated with a loss of cells from the IC outlet port of thebioreactor.

Process 2100 next proceeds to query 2128, in which it is determinedwhether to continue to add reagent or other bolus addition on other daysor other time intervals, for example. If it is desired to add additionalreagent or other bolus and re-distribute cells, process 2100 branches“yes” to add reagent and re-distribute cells 2118. For example, suchbolus addition and re-distribution of cells may next occur on Days 6 and9, according to embodiments.

If, or once, it is not desired to continue adding a bolus, e.g.,reagent, and re-distributing the cells, process 2100 proceeds “no” toharvest the cells 2130, in which the cells may be transferred to aharvest bag(s) or container(s). Process 2100 then terminates at ENDoperation 2136.

Alternatively, from harvest operation 2130, process 2100 may optionallyproceed to allow for further processing/analysis 2132. Such furtherprocessing 2132 may include characterization of the phenotype(s), forexample, of the harvested cells, e.g., T cells or Tregs. From optionalfurther processing/analysis step 2132, process 2100 may proceed tooptionally reload any remaining cells 2134. Process 2100 may thenterminate at END operation 2136.

Process 2200 illustrates operational steps for a process of expandingcells in cell expansion system according to embodiments of the presentdisclosure. Process 2200 may be used in some embodiments to expand Tcells. As illustrated, various steps may be performed over the course ofa 14-day protocol to expand the cells. START operation 2202 is initiatedand process 2200 proceeds to Day 0, where a disposable set is loadedonto a cell expansion system 2206. The disposable set may then be primed2208, in which the set may be primed 2208 with PBS (e.g., LonzaCa2+/Mg2+−free), for example. In preparation for the loading of cells,the priming fluid may be exchanged using an IC/EC washout 2210. Forexample, the PBS in the system may be exchanged for TexMACS GMP BaseMedium, according to one embodiment. The media may next be conditioned2212. The condition media 2212 may be performed to allow the media toreach equilibrium with provided gas supply before cells are loaded intoa bioreactor.

Next on Day 0, process 2200 proceeds to load cells 2214, e.g.,suspension or non-adherent cells, such as T cells or Tregs. In anembodiment, such cells may be loaded 2214 by a “load cells centrallywithout circulation” task. In another embodiment, such cells may beloaded 2214 by a “load cells with uniform suspension” task.

At Day 3 2216, a bolus of cytokines may be added while the cells areredistributed 2218. In embodiments, the redistribution of cells may beperformed in combination with the bolus addition to mix the cells andmore thoroughly expose the cells to the cytokines (e.g., IL-2) that maybe in the bolus addition. In embodiments, the redistribution may alsobreak up colonies or clusters of cells that may have formed. Inembodiments, the redistribution may occur first by circulating the cellsin a fluid circulation path. The bolus addition may then be added in theprocess of pushing the cells back into the bioreactor, such as byintroducing fluid into a fluid circulation path to push cells back intothe bioreactor. After the redistribution and bolus addition 2218,process 2000 proceeds to feed cells 2220.

The cells may again be redistributed 2224 with another bolus addition atDay 6 2222. The redistribution may break up colonies or clusters ofcells that may have formed during Days 3-5. The bolus addition mayexpose the cells to additional reagents that promote expansion. Process2000 proceeds to feed cells 2226 at Day 6. At Day 9 2228, the cells mayonce again be redistributed 2230 with a bolus addition. Theredistribution may break up colonies or clusters of cells that may haveformed during Days 6-8. The bolus addition may expose the cells toadditional supplements that promotes expansion. Process 2000 proceeds tofeed cells 2232 at Day 9.

At Day 11-13 2234, the cells may again be redistributed 2236 with abolus addition. The redistribution may break up colonies or clusters ofcells that may have formed during Days 9-10. The bolus addition mayexpose the cells to additional reagents that promote expansion. Process2000 then proceeds to feed cells 2238. In embodiments, the steps 2236and 2238 may be performed on each of Day 11, Day 12, and Day 13. Thismay occur as a result of the cells expanding during Days 0-10 and therebeing larger numbers of cells in the bioreactor. Performing theredistribution and bolus addition of cells may promote expansion of thecells by breaking up colonies and clusters of cells more often andmixing them with reagents in the bolus addition to promote cellexpansion. Process 2200 terminates at END operation 2240.

Process 2300 illustrates operational steps for a process of expandingcells, e.g., suspension or non-adherent cells, in a cell expansionsystem according to embodiments of the present disclosure. Process 2300may be used in some embodiments to expand T cells, such as Tregs. Thecombination of steps of process 2300 may allow the expansion of thecells to useful clinical amounts using initial low seeding densities.

START operation 2302 is initiated and process 2300 proceeds to loaddisposable set 2304 onto a cell expansion system. The disposable set maythen be primed 2206, in which the set may be primed 2206 with PBS (e.g.,Lonza Ca2+/Mg2+−free), for example. In preparation for the loading ofcells, the priming fluid may be exchanged using an IC/EC washout 2308.For example, the PBS in the system may be exchanged for TexMACS GMP BaseMedium, according to one embodiment. The media may next be conditioned2310. The condition media 2310 may be performed to allow the media toreach equilibrium with a provided gas supply before cells are loadedinto a bioreactor.

Process 2300 proceeds to 2312 load inlet volume of fluid with cells. Inembodiments, the cells may comprise non-adherent cells, such as one ormore types of T cells, e.g., Tregs. In one embodiment, the cellscomprise Tregs. Embodiments may provide for the inlet volume of fluidwith the cells to be loaded through an IC inlet path utilizing an ICinlet pump and into an IC circulation path. In embodiments, the loadinlet volume 2312 is loaded without activating an IC circulation pump.

Process 2300 proceeds to positioning the inlet volume in a first portionof a bioreactor 2314. In embodiments, the positioning may be performedby introducing a second volume of fluid, which may comprise media andmay be introduced into a portion of the IC circulation path to push theinlet volume with the cells into the first position in the bioreactor.In embodiments, the inlet volume of fluid and the second volume of fluidmay be the same. In other embodiments, the inlet volume of fluid and thesecond volume of fluid may be different. In yet other embodiments, a sumof the inlet volume of fluid and the second volume of fluid may be equalto a percentage of a volume of the IC circulation path.

Following the position inlet volume 2314, process 2300 proceeds toexpose cells to activator 2316 in order to activate the cells to expand.The cells may be exposed to an activator 2316 that is soluble in someembodiments. The activator, which may include antibody complexes in someembodiments, may be added to the media and may be included in the inletvolume or added later, such as with the second volume. In embodiments,the activator may be a human antibody CD3/CD28/CD2 cell activatorcomplex, for example.

Process 2300 proceeds to feed the cells per a first process during afirst time period 2318. In an embodiment, the cells may be fed at aminimum or low feed rate, for example, where the cell population isbeginning to grow/expand for a first time period 2320, and a minimum orlow feed rate is able to meet the demands of such population. Forexample, an IC inlet pump rate of +0.1 mL/min may be used during suchfirst time period. If it is desired to reduce the loss of cells from thehollow fiber membrane bioreactor during such first time period, the ICinlet pump rate of +0.1 mL/min may be matched, or closely orsubstantially matched, to a complementary IC circulation pump rate of−0.1 mL/min to maintain cells in the bioreactor during the growth phaseof the cell culture.

From 2320, process 2300 proceeds to expand cells during a second timeperiod 2322. Expanding during the second period of time 2322 may alsoinvolve feeding the cells per a second process during the second timeperiod 2324. In an embodiment, such second process may involve feedingthe cells at substantially the same feed rates as during the first timeperiod, for example. In another embodiment, the second process mayinvolve feeding the cells at different feed rates as compared to thefeed rates used during the first time period. For example, the feedrates may increase as a result of the expansion of the cells during thefirst time period.

While expanding the cells during the second time period 2322, the cellsmay also be circulated to shear cells colonies or cell clusters 2326.Step 2326 may involve circulating the cells in the IC circulation pathto shear any colonies or clusters that may have formed during the firsttime period. The shear colonies or clusters 2326 step may reduce anumber of cells in a cell colony or cell cluster. In embodiments, thecirculate to shear 2326 may cause cell colonies to incur a shear stress,causing one or more cells in the cell colony to break apart from thecell colony.

Process 2300 may next proceed to harvest operation 2328, in which thecells may be transferred to a harvest bag(s) or container(s). Inembodiments, a therapeutic dose of cells may be harvested. Inembodiments, the cells harvested at operation 2328 may be on the orderof 1×10⁹ cells. The harvested cells may have viabilities between about75% and about 95%, in embodiments.

Process 2300 may then optionally proceed to allow for furtherprocessing/analysis 2330. Such further processing may includecharacterization of the phenotype(s), for example, of the harvestedcells, e.g., T cells or Tregs. In one embodiment, the harvested cellsmay express biomarkers consistent with Tregs. For example, the cells mayexpress CD4+, CD25+, and/or FoxP3+ biomarkers. In embodiments, theharvested cells may include the CD4+CD25+ phenotype at a frequency ofabove about 80%. In other embodiments, the cells may include theCD4+FoxP3+ phenotype at a frequency of above about 55%. Process 2300 maythen terminate at END operation 2332.

The operational steps depicted in the above figures are offered forpurposes of illustration and may be rearranged, combined into othersteps, used in parallel with other steps, etc., according to embodimentsof the present disclosure. Fewer or additional steps may be used inembodiments without departing from the spirit and scope of the presentdisclosure. Also, steps (and any sub-steps), such as priming,conditioning media, loading cells, for example, may be performedautomatically in some embodiments, such as by a processor executingpre-programmed tasks stored in memory, in which such steps are providedmerely for illustrative purposes. Further, the example pump ratesettings for feeding cells depicted in FIG. 10B, for example, areoffered for purposes of illustration. Other pump rates, flow rates,directions, etc. may be used in accordance with embodiments of thepresent disclosure.

Examples and further description of tasks and protocols, includingcustom tasks and pre-programmed tasks, for use with a cell expansionsystem are provided in U.S. patent application Ser. No. 13/269,323(“Configurable Methods and Systems of Growing and Harvesting Cells in aHollow Fiber Bioreactor System,” filed Oct. 7, 2011) and U.S. patentapplication Ser. No. 13/269,351 (“Customizable Methods and Systems ofGrowing and Harvesting Cells in a Hollow Fiber Bioreactor System,” filedOct. 7, 2011), which are hereby incorporated by reference herein intheir entireties for all that they teach and for all purposes.

Next, FIG. 24 illustrates example components of a computing system 2400upon which embodiments of the present disclosure may be implemented.Computing system 2400 may be used in embodiments, for example, where acell expansion system uses a processor to execute tasks, such as customtasks or pre-programmed tasks performed as part of processes such asprocesses illustrated and/or described herein. In embodiments,pre-programmed tasks may include, follow “IC/EC Washout” and/or “FeedCells,” for example.

The computing system 2400 may include a user interface 2402, aprocessing system 2404, and/or storage 2406. The user interface 2402 mayinclude output device(s) 2408, and/or input device(s) 2410 as understoodby a person of skill in the art. Output device(s) 2408 may include oneor more touch screens, in which the touch screen may comprise a displayarea for providing one or more application windows. The touch screen mayalso be an input device 2410 that may receive and/or capture physicaltouch events from a user or operator, for example. The touch screen maycomprise a liquid crystal display (LCD) having a capacitance structurethat allows the processing system 2404 to deduce the location(s) oftouch event(s), as understood by those of skill in the art. Theprocessing system 2404 may then map the location of touch events to UIelements rendered in predetermined locations of an application window.The touch screen may also receive touch events through one or more otherelectronic structures, according to embodiments. Other output devices2408 may include a printer, speaker, etc. Other input devices 2410 mayinclude a keyboard, other touch input devices, mouse, voice inputdevice, etc., as understood by a person of skill in the art.

Processing system 2404 may include a processing unit 2412 and/or amemory 2414, according to embodiments of the present disclosure. Theprocessing unit 2412 may be a general purpose processor operable toexecute instructions stored in memory 2414. Processing unit 2412 mayinclude a single processor or multiple processors, according toembodiments. Further, in embodiments, each processor may be a multi-coreprocessor having one or more cores to read and execute separateinstructions. The processors may include general purpose processors,application specific integrated circuits (ASICs), field programmablegate arrays (FPGAs), other integrated circuits, etc., as understood by aperson of skill in the art.

The memory 2414 may include any short-term or long-term storage for dataand/or processor executable instructions, according to embodiments. Thememory 2414 may include, for example, Random Access Memory (RAM),Read-Only Memory (ROM), or Electrically Erasable Programmable Read-OnlyMemory (EEPROM), as understood by a person of skill in the art. Otherstorage media may include, for example, CD-ROM, tape, digital versatiledisks (DVD) or other optical storage, tape, magnetic disk storage,magnetic tape, other magnetic storage devices, etc., as understood by aperson of skill in the art.

Storage 2406 may be any long-term data storage device or component.Storage 2406 may include one or more of the systems described inconjunction with the memory 2414, according to embodiments. The storage2406 may be permanent or removable. In embodiments, storage 2406 storesdata generated or provided by the processing system 2404.

EXAMPLES

The following description includes some examples ofprotocols/methods/processes that may be used with a cell expansionsystem, such as CES 500 (e.g., FIGS. 5A, 5B, 5C) and/or CES 600 (FIG.6), for example, that implements aspects of the embodiments. Althoughspecific features may be described in the examples, such examples areprovided merely for illustrative and descriptive purposes. For example,while examples may provide for the expansion of T cells and/or Tregcells, other and/or additional cell types and/or combinations thereofmay be used in other embodiments. Although specific parameters,features, and/or values are described, e.g., use of a CES, e.g.,Quantum® Cell Expansion System, according to some embodiments, theseparameters, features, and/or values, etc., are provided merely forillustrative purposes. The present disclosure is not limited to theexamples and/or specific details provided herein.

Further, the examples provided herein are not intended to limit otherembodiments, which may include different or additional steps,parameters, or other features. The example methods or protocols,including the steps (and any sub-steps), may be performed automaticallyin some embodiments, such as by a processor executing pre-programmedtasks stored in memory. In other embodiments, the steps (and anysub-steps) may be performed through the combination of automated andmanual execution of operations. In further embodiments, the steps (andany sub-steps) may be performed by an operator(s) or user(s) or throughother manual means.

While example data may be provided in such examples, such example dataare provided for illustrative purposes and are not intended to limitother embodiments, which may include different steps, parameters,values, materials, or other features.

Example 1

Methods

General Treg Cell Culture

Immunomagnetic-isolated CD4⁺CD25⁺ Tregs may be acquired from healthyadult donor peripheral blood by leukapheresis (HemaCare Corporation, VanNuys, Calif.) and may be subsequently expanded at Terumo BCT at aconcentration of 1.0×10⁵ cells/mL in sterile-filtered TexMACS™ GMPMedium supplemented using three T25 flasks (7 mL/flask) with therecombinant human IL-2 IS Premium grade cytokine at 200 IU/mL (MiltenyiBiotec GmbH, Bergisch Gladbach) and Gibco PSN 100X antibiotic mixture(ThermoFisher Scientific, Waltham, Mass.). The actively growing Tregcell suspension may be subsequently used as the inoculum in each of thethree (3) Quantum Cell Expansion System experimental runs. Tregs forboth the inoculum and the Quantum System expansion may be co-stimulatedusing a soluble tetrameric Immunocult™ human antibody CD3/CD28/CD2 cellactivator complex (Stem Cell Technologies, Vancouver, BC) at 25 μL/mL inthe absence of microbeads. Co-stimulation may be performed on Days 0 and9 for the Treg inoculum and on Day 0 for the Quantum System Tregexpansion. The Quantum System HFM bioreactor may be characterized by anintracapillary loop volume of 177.1 mL and surface area of 21,000 cm².

Quantum System Treg Expansion

According to embodiments, two (2 L) bags of sterile filtered media maybe prepared for the Treg scale up expansion in the Quantum System usingthe Quantum Media Bag 4 L Set (Cat. 21021). One 2 L bag of completemedia containing TexMACS GMP, IL-2 and PSN antibiotics may be used tosupply the IC compartment and one 2 L of base media containing TexMACSGMP and PSN antibiotics may be used to supply the EC inlet compartmentof the bioreactor. After priming the Quantum System with PBS (Lonza Cat.17-516Q, Walkersville, Md.), media bags may be connected to theappropriate IC and EC inlet lines using the TSCD-Q Terumo SterileWelder. The complete media may be protected from exposure to light.

The total cell load for each run (4.5-6.5×10⁷ Tregs) may be resuspended,using aseptic technique, in 50 mL of complete medium with a Quantum CellInlet Bag (Cat. 21020) for introduction into the Quantum Systembioreactor. Additional disposable bags, such as the Quantum CES MediaBag 4 L (Cat. 21021) and Waste Bag 4 L (Cat. 21023) may also be usedduring the Treg scale-up expansion runs.

At the completion of the “Load Cells Centrally without Circulation”Task, the Quantum System runs (n=3) may be seeded with Tregs at aconcentration of 2.5-3.7×10⁵ cells/mL in 177 mL of complete medium or anaverage of 2.1-3.1×10³ cells/cm² within the lumen or the intracapillary(IC) compartment of the hollow fiber membrane bioreactor.

Day 0-4:

Example Quantum Custom Task

Feed Cells, Modified.

IC/EC Exchange & Condition Media for Regulatory T Cells, Example.

Attach the TexMACS GMP Complete Medium with IL-2 supplement (200 IU/mL)Media bag to the IC Media line of the Quantum System with the Terumo BCTTSCD-Q sterile welder. Attach TexMACS Base Media to EC Media line.Perform IC/EC Washout and Condition Media Tasks respectively. CompleteMedia may be used for IC Exchange or Washout and Base Media may be usedfor the EC Exchange or Washout to conserve the amount of IL-2 andactivator complex.

Place system on modified “Feed Cells” prior to introducing cells.Increase the IC inlet rate (Q₁) and IC circulation rate (Q₂) in amatched rate to 0.2, 0.3, and 0.4 mL/min on Days 5, 6, and 7, butopposite direction on Days 4, 5, 6 or as needed to keep the lactatelevel between 5-8 mmol/L.

TABLE 1 Feed Cells, Modified, Example. Table 1: Feed Cells Setting Step1 IC Inlet IC Media IC Inlet Rate 0.1 IC Circulation Rate −0.1   ECInlet None EC Inlet Rate  0.00 EC Circulation Rate 100     Outlet ECWaste Rocker Control No Motion: ( 0° ) Stop Condition Manual EstimatedFluid Unknown Omit or Include Include

Example Quantum Custom Task:

Load Cells Centrally without Circulation, Example.

Purpose: This task enables suspension cells to be centrally distributedwithin the bioreactor membrane while allowing flow on the extracapillary(EC) circulation loop. The pump flow rate to the IC loop may be set tozero.

Prior to loading the cells into the Quantum systems using the Load Cellswithout Circulation, enter the modifications to the task.

TABLE 2 Load Cells Centrally without Circulation, Modifications, ExampleTable 2: Solutions for Loading Suspension Cells Volume (estimate basedon factory Bag Solution in Bag default values) Cell Inlet 50 mL N/AReagent None N/A IC Media Serum-Free Media 6 mL/hour Wash None N/A ECMedia None N/A

TABLE 3 Load Cells Centrally without Circulation, Example Table 3:Custom Task 8 Load Cells without Circulation Setting Step 1 Step 2 ICInlet Cell IC Media IC Inlet Rate 50 50 IC Circulation Rate   0   0 ECInlet None None EC Inlet Rate    0.00    0.00 EC Circulation Rate 30 30Outlet IC Waste IC Waste Rocker Control In Motion: In Motion: (−90 to 180° ) ( −90 to 180° ) Dwell Time Dwell Time 1 sec 1 sec StopCondition Empty Bag IC Volume: 57.1 mL Estimated Fluid Unknown <0.1 LOmit or Include Include Include

Return to default cell feeding tasks as needed and continue with theexpansion protocol using Feed Cells Task.

TABLE 4 Feed Cells, Modified, Example. Table 4: Feed Cells Setting Step1 IC Inlet IC Media IC Inlet Rate 0.1 IC Circulation Rate −0.1   ECInlet None EC Inlet Rate  0.00 EC Circulation Rate 100     Outlet ECWaste Rocker Control No Motion: ( 0° ) Stop Condition Manual EstimatedFluid Unknown Omit or Include Include

Day 4 or Later:

Resuspension of Treg Cells During Cell Culture or Prior to Harvest,Example.

A purpose of this modified Circulation Task is to uniformly resuspendthose cells that may be loosely adhered during culture or prior toinitiating the Harvest Task.

In addition, this task may be used to shear Treg cell colonies every two(2) days during cell culture in order to maintain uniform cell densityand nutrient diffusion beginning on or after Day 4. If the task is usedto shear colonies during the culture process, the Quantum System may bereturned to the modified “Feed Cells” Task.

TABLE 5 Circulation and Resuspension of Cells, Return Cells toBioreactor, & Feed, Example Table 5: Custom Task 6 Settings to ResuspendSettled Cells Setting Step 1 Step 2 Step 3 Step 4 IC Inlet None IC MediaIC Media IC Media IC Inlet Rate   0 0.1 100 0.1 IC Circulation Rate 300−0.1   −70 −0.1   EC Inlet None None None None EC Inlet Rate   0 0     0 0    EC Circulation Rate 100 100     100 100     Outlet EC OutletEC Outlet EC Outlet EC Outlet Rocker Control In Motion: StationaryIn Motion: In Motion: ( −90 to 180° ) ( −90 to 180° ) ( −90 to 180° )Dwell Time: Dwell Time: Dwell Time: 1 sec 1 sec 1 sec Stop ConditionTime: Time: IC Volume Manual 4 min 1 min 150 mL Estimated Fluid Unknown0.1 L 0.2 L Unknown Omit or Include Include Include Include Include

Harvest Quantum Harvest Task with Modification, Example.

TABLE 6 Harvest, Modification, Example Table 6: Harvest, ModifiedSetting Step 1 Step 2 IC Inlet None IC Media IC Inlet Rate   0 400  ICCirculation Rate 300 −70  EC Inlet None IC Media EC Inlet Rate   0 60 ECCirculation Rate 100 30 Outlet EC Outlet Harvest Rocker ControlIn Motion: In Motion: ( −90° to 180°, ) (−90° to 180°) Dwell Time: DwellTime: 1 sec 1 sec Stop Condition Time: IC Volume: 4 minutes 378 mLEstimated Fluid IC Media: <0.1 L IC Media: 0.5 L Omit or Include IncludeInclude

Harvested cells may be removed from the Quantum System by RF welding forfurther evaluation and analysis.

Post-Harvest Analysis

Harvested cells may be enumerated with a Vi-CELL XR 2.04 Cell ViabilityAnalyzer (Beckman Coulter) over a range of 5-50 μm and quantified formembrane integrity by trypan blue dye exclusion.

Metabolism

Regulatory T cell metabolism may be monitored from the Quantum EC sampleport daily by i-STAT handheld analyzer (Abbott Point of Care, Princeton,N.J.) using G Cartridge for glucose and lactate concentrations usingi-STAT G Cartridge (Cat. 03P83-25) and i-STAT CG4+ Cartridge (Cat.03P85-50) respectively.

Cell Surface Biomarker Expression

Human Regulatory T cells (natural and induced) compose a small subset(2-10%) of all T cells in human cord blood and peripheral blood.Functionally, Tregs are responsible for maintaining immunologicalhomeostasis which includes the modulation of immune tolerance in bothinnate and adoptive responses. Moreover, the expression of thetranscriptional regulator forkhead box P3 (FoxP3) gene product is knownto correlate with the CD4⁺CD25⁺FoxP3⁺CD127^(lo/−) Treg phenotype and theimmune suppression of antigen presentation cells (APCs) and effector Tcells (T_(eff)). IL-2 binding to the CD25/IL-2 receptor (Rα) and theactivation of STATS transcriptional factor is used for Foxp3 induction.FoxP3 suppression upregulates the activity of several genes such asCTLA-4, TNFRSF18 and IL2RA and downregulates IL-2 via its associationwith histone acetylase KAT5 and histone deacetylase HDAC7.

Treg phenotype frequency of the harvested cell surface biomarkers may bequantified by flow cytometry. To this end, the cells may be stained withthe following antibody conjugates and gated against viable, unstainedcells: Fixable Viability Dye eFluor® 780 (eBioscience 65-0865), mouseanti-human CD4-PE (BD Pharmingen 561844), anti-CD4-Alexa Fluor 647 (BDPharmingen 557707), anti-CD4-FITC (BD Pharmingen 561842), anti-CD4-FITC(BD Pharmingen 555346), anti-CD25-PE (BD Pharmingen 555432),anti-CD127-PE (BD Pharmingen 557938), anti-CD45RO-PE (BD Pharmingen347967), and anti-FoxP3-Alexa Fluor 647 (BD Pharmingen 560045). Specimendata may be acquired on a bead-compensated BD Canto II flow cytometerequipped with FACSDiva v6.1.3 software using 1×106 cells and 20,000total events per sample.

Experimental Flow, Example

Possible Results

Preliminary studies with Tregs in static culture may show that thesecells tend to form micro-colonies on the order of 100 μm in diameter.Separating these cells during the medium exchange process every two daysmay help to limit cellular necrosis and return the cells to a highdensity, single cell suspension by using a 1,000 μL pipet tip that hasan ID of 762 μm. Alternatively, the process of maintaining a single cellsuspension may be accomplished more efficiently in an automated HFMbioreactor where the fiber lumen ID is on the order of 200 μm, such asin the Quantum System, with the aid of a preprogrammed, dailycirculation task. In addition, this automated feeding task may reducethe likelihood of contamination while maintaining continuous nutrientflow to the Treg culture since it may be performed in a functionallyclosed system.

Possible Treg Cell Density and Viability

TABLE 7 T25 Flask Possible Harvest Cell Density 3.11 × 10⁶ cells/mL 8.71 × 10⁵ cells/cm2 Viability 81.90% Stimulation Cycles 2   Doublings4.9

TABLE 8 Quantum Possible Harvest Cell Density Harvest Bag 3.61 × 10⁶cells/m  Bioreactor  1.24 × 10⁷ cells/mL ≥7.33 × 10⁴ cells/cm2 Viability84.80% Stimulation Cycles 1    Doublings 4.6  Doubling Time 41.6 hours

Preliminary Cell Seeding Density Experiments

In preparation for the expansion of immunomagnetic selected cells fromthe Donor in the automated bioreactor, a series of static growthexperiments may be performed to determine if stimulated Tregs may becultured at a seeding density of less than 1.0×10⁶ cells/mL. Thisportion of the study may be performed by seeding 18 wells of a 24-welltissue culture plate 1.0×10⁵ cells/mL or well in TexMACS GMP mediumsupplemented with IL-2 (200 IU/mL) and PSN antibiotics. These cells mayalso be co-stimulated with the soluble anti-CD3/CD28/CD2 mAb complex onDay 0 and Day 9 at 25 μL/mL. Cells may be manually harvested and countedon Day 14 by Vi-CELL XR.

TABLE 9 Summary of possible Treg cell seeding static plate test. AverageTreg Plate Average Treg Average Treg Treg Samples Seeding HarvestHarvest Treg Plate Harvest (1 mL Day 0 Day 14 Day14 Treg Cell Treg CellCell Viability each) (viable cells/mL) (viable cells/mL) (cells/cm²) DSDT (hours) (Trypan Blue Exclusion) n = 18 1.00 × 10⁵ 2.65 × 10⁶ 1.33 ×10⁶ 4.7 71.0 61.5% SE 4.14 × 10⁴ Abbreviations: Doublings (DS), DoublingTime (DT).

After harvest, the cell samples may be pooled for Treg biomarkeranalysis by flow cytometry. Possible results may show that thefrequencies of CD4⁺C25⁺, CD4⁺CD127⁻, and CD4⁺FoxP3⁺ phenotype may berespectively 90.9%, 79.7%, and 31.6% in static culture. The CD4⁺CD25⁺phenotype (>70%) is generally the most reliable determinant for Tregbiomarker identification since FoxP3+ detection is highly dependent onthe permeabilization method and cell viability.

The data from this static plate test may suggest that human Tregs may beexpanded, with cell seeding densities on the order of 10⁵ cells/mL, whencultured in the presence of a soluble co-stimulation anti-CD3/CD28/CD2mAb complex and serum-free medium.

Treg Metabolism

Regulatory T cells are dependent on mitochondrial metabolism and havethe ability to oxidize multiple carbon sources, i.e., lipid or glucose.Tregs are known to shift their metabolism from fatty acid oxidation(FAO) to glycolysis when they enter a highly proliferative state as aresult of mTOR regulation of glycolysis and fatty acid metabolism.Moreover, it has been shown that glycolysis may be necessary for thegeneration and suppressive functionality of human inducible Tregs bymodulating the expression of FoxP3 variants through IL-2/STATS/enolase-1promoter signaling and 2-Deoxy-D-glucose inhibition studies.Accordingly, monitoring the glucose and lactate levels may facilitatethe adjustment of Quantum System media flow rates to support Tregexpansion in the hollow fiber bioreactor. Initially, the Tregs may bethought to transiently reduce their metabolic rate before they enter thecell cycle and proliferate. This may be supported by the transientreduction of glycolysis and mTOR activity in freshly isolated humanTregs before TCR stimulation. Specifically, mTORC1 may be thought toincrease the expression of glucose transporters such as Glut-1-mediatedglucose transport as a consequence of the upregulated mTOR pathway.

The possible results of three (3) expansions from three separate Tregcell aliquots may indicate that the glucose consumption and lactategenerate may appear to correlate within each Quantum System run. Allthree of the ex vivo expansion runs may show that glucose consumption inTregs may increase above background levels by Day 1 and 2 out of 3 runsmay show that the lactate generation levels may increase abovebackground levels by Day 2. One run, with reduced cell viability atthaw, may generate a lagging lactate generation rate which may bereflected in the cell harvest yield. Maximum glucose consumption ratesfor the 2 out of 3 runs, in the most actively growing Treg cultures, maybe 1.618 and 2.342 mmol/day on Days 8 and 7 respectively. The maximumlactate generation rates may be 2.406 and 3.156 mmol/day at the sametime points.

Throughout the Treg expansion runs, an effort may be made to control thelactate values at 7 mmol/L by concurrently increasing both the IC Input(+) and IC circulation (−) pump rates from (±0.1 to ±0.4 mL/min) withinthe lumen of the hollow fiber membrane over Days 4-8. The lowest glucoselevels during the course of the Treg cell expansions may range from 264mg/dL on Day 7 (Q1584) to 279 mg/dL on Day 8 (Q1558). The base glucoseconcentration, in the cell growth formulated medium for thesefeasibility expansions, may be 325 to 335 mg/dL which may be found to besupportive when used in conjunction with the Quantum System flow rateadjustments.

Regulatory T Cell Biomarker Expression

The evaluation of the Treg cell harvest by flow cytometry may becentered on the CD4⁺CD25⁺FoxP3⁺ T cell subsets in this feasibilitystudy. In T lymphocytes, the human CD4 gene, on Chromosome 12, encodesfor a membrane glycoprotein which interacts with the majorhistocompatibility complex class II and functions to initiate the earlyphase of T cell activation. In regulatory T cells, the human CD25 (IL2R)gene, on Chromosome 10, encodes for the IL-2 receptor and functions bysequestering the cytokine IL-2. In regulatory T cells, theforkhead/winged-helix box P3 human gene, on the Chromosome X, encodesfor the FoxP3 transcriptional factor which may be essential for Tregsuppressor function. FoxP3 gene product binds to the promoter region ofCD25, CTLA-4 and IL-2, IL7R, IFN-γ genes thereby upregulating CD25 andCTLA-4 and repressing IL-2, IL7R, and IFN-γ gene transcription. TheCD127 gene encodes for the IL-7 receptor and Tregs may be generallycharacterized by low CD127 (IL-7R) expression when compared toconventional T cells. However, certain Treg subsets are known to expresshigh CD127 levels during in vitro and in vivo activation which maycorrelate to a higher Treg survival when the cells are incubated withIL-7. The CD45RO gene product is expressed on naive thymus derived Tregsthat upon activation lose CD45RA and express CD45RO.

TABLE 10 Possible regulatory T cell biomarker expression as a percent ofparent population. Quantum System Seeding Harvest Expansion RunViability Viability CD4⁺CD45RO⁺ CD4⁺CD25⁺ CD4⁺CD127^(low) CD4⁺FoxP3⁺Q1558 81.9% 84.8% 72.4% 86.7% 40.1% 58.2% Q1567* 49.6% 69.8% 55.3% 79.3%74.2% *5.6% Q1584 90.7% 94.6% 72.8% 90.5% 41.0% 64.9% Average (n = 3)85.5% *42.9% Average (n = 2)* *61.6% *Low cell viability at thaw/harvestand incomplete permeabilization on Q1567 cells for FoxP3+ frequency.

The average expression of the CD4⁺CD25⁺ Treg phenotype frequency may be85.5% in the cells harvested from the Quantum System which may comparefavorably with the published CD4⁺CD25⁺ release criteria of >70%. In the01567 Treg expansion, the elevated frequency of the CD4⁺CD127^(low)population (74.2%) may be a reflection of the low cell viability in thisparticular thawed cell sample since these cells may be cultured onlywith IL-2 as a cytokine supplement, according to an embodiment. In cellsexpanded by the two Quantum System runs with seeding and harvestviability above 80%, the CD4⁺FoxP3⁺ expression frequency may be 61.6%.This finding may be consistent with the published release specificationof 60% for FoxP3⁺. Furthermore, the results of the two billion cellexpansions may compare favorably with the CD3⁺CD45⁺ (87.30%), CD25⁺(47.76%), and FoxP3⁺ (59.64%) biomarker expression in the original donorTreg cell specimen which may be received from HemaCare BioResearchProducts.

Additional flow cytometry analysis may be performed on cryopreservedTreg cells from the 01584 expansion run by a third-party laboratory, forexample, using fluorescence Minus One (FMO) gating, different stains,and different instrumentation. FMO control is a type of gating controluse to interpret cell populations by taking into account the spread ofall the fluorochromes in the data plots minus the one used to quantifythe frequency of a particular marker. For example, the flow results fromthe third-party laboratory may indicate that the CD4⁺CD25⁺ Treg cellpopulation frequency may be 95.4% from the 01584 run which may comparefavorably with the 90.5% which may be found by the Terumo BCT CESLaboratory. Incomplete staining with the alternative anti-FoxP3-PE clonestain may limit the third-party laboratory quantification of thisinternal biomarker, but the dot-plots may suggest that there may be asubpopulation of high expressing FoxP3⁺ Tregs in the 01584 specimen thatmay not be observed in the Control Treg cell reference sample. Althoughinteresting, additional studies may be needed to confirm theseobservations.

Harvest Yield

The possible average diameter of viable (trypan blue exclusion) Tregcells at Quantum System harvest may be 10.89, 11.04, and 11.06 μmrespectively across the Q1558, Q1567, and Q1584 runs over a range of5-50 μm as defined with 50 samples for each run. This may compare to anaverage cell diameter of 11.91, 12.40, and 7.83 μm respectively fromflasks at the time of bioreactor seeding.

These possible cell diameter data may suggest that there may be moreuniformity in the diameter of the cells harvested from the QuantumSystem than there may be in the diameter of the cells which may beexpanded in the inoculum flasks.

The Treg Quantum System possible harvest data are summarized in Table11. Moreover, the impact of the CD4⁺CD25⁺ cell viability at the point ofseeding the bioreactor may be evident when comparing the results ofQ1554/1584 harvests with the Q1567 harvest. There may be a 32-41% higherviability in the bioreactor inoculum for the Q1554/1584 expansion runsversus the viability for the Q1567 run. This may be due to a variationin the original cell isolation, cryopreservation technique or the lengthof storage since the cell aliquots that may be used in this study(HemaCare PB425C-2; Lot 14034019) may be derived from the same donorcollection on Feb. 11, 2014.

TABLE 11 Possible expansion of Treg cells from inoculum flasks toQuantum System harvests. Treg Treg Treg Treg Viability Treg ViabilityTregs DS (11 Days) DS (7-8 Days) DT (hours) Quantum Flask QuantumQuantum Flask Quantum Quantum Run Inoculum^(A) Harvest^(A) Harvest^(A)Inoculum^(B) Harvest^(B) Harvest^(B) Q1554 81.9% 84.8% 1.82 × 10⁹ 5.04.8 38.5 Q1567 49.6% 69.8% 1.59 × 10⁸ 4.4 1.8 101.3 Q1584 90.7% 94.6%1.30 × 10⁹ 4.7 4.6 35.7 Abbreviations: DS—Population Doublings,DT—Population Doubling Time in hours. ^(A)Harvest data may be based onVi-Cell XR counts with Trypan Blue for membrane integrity. ^(B)Note: theTreg cell inoculum from flasks may receive two (2) rounds costimulationon Days −0 and −9; whereas, the Tregs which may be harvested from theQuantum Systems may receive one (1) round of costimulation on Day-0.

The objective of this feasibility study may be to determine if theQuantum System may support the expansion of Tregs in the range of7.0×10⁷-1.4×10⁹ cells with commercially available supplements. Two ofthe three bioreactor harvests from Q1554 and Q1584 may generate anaverage of 1.56×10⁹ Tregs, using a soluble anti-CD3/CD28/CD2 mAbco-stimulator complex, from a seeding density of <1.0×10⁶ cells/mL inless than eight (8) days. This may translate into an average harvestcell density of 8.81×10⁶ cells/mL or 7.43×10⁴ cells/cm² in the IC loopof the Quantum System bioreactor over the Q1554/1584 runs.

Possible Conclusions

The results of this feasibility study may be exploratory in nature andmay not necessarily be designed to cover all technical options. Forexample, one could consider the reduction of inoculum Tregco-stimulation from two (2) to one (1) activation events. As such, themethods which may be used in the automated Quantum System expansion ofimmunomagnetic-isolated regulatory T cells may be open to modification.Our attempt here may be to define certain technical aspects of theculture process that may be conducive to further study in the upscaleexpansion of Tregs within the Quantum System platform. Within thiscontext, the possible study findings may suggest that these possibleconclusions or observations may be reasonable and may be helpful in theproduction of regulatory T cells for research, development, orproduction purposes.

Human Tregs, as identified as FoxP3⁺/CD25⁺, may be cultured and may beexpanded with a soluble co-stimulatory anti-CD3/CD28/CD2 monoclonalantibody (mAb) T cell complex, in the absence of co-stimulatorymAb-coated beads, when supplemented with the cytokine IL-2 in theQuantum System automated hollow fiber bioreactor.

Human Tregs may be efficiently expanded in the Quantum system from cellseeding densities of less than 1×10⁶ cells/mL or less than 6.6×10⁴cells/cm². To this end, the objective of harvesting Tregs within therange of 7.0×10⁸ to 1.4×10⁹ cells in less than 14 days may be achievedwith an average (n=3) of 1.09×10⁹ total cells. An average of 85.5% ofthe cells may express the Treg CD4⁺CD25⁺ phenotype and an average of42.9% may express CD4+FoxP3+ phenotype (n=3). In the two (2) billioncell Quantum System expansions, an average of 61.6% of the total cellsmay express the CD4⁺FoxP3⁺ phenotype. One of the three Quantum systemTreg cell expansion runs may be validated for CD4⁺CD25⁺ expression by athird-party laboratory human IMSR due to the limited number of cells.

Human Tregs may be successfully cultured and may be expanded in theQuantum System by centrally seeding the cells within the lumen (IC loop)of an automated hollow fiber bioreactor.

Media IC input (+0.1 to +0.4 mL/min) and IC circulation (−0.0 to −0.4mL/min) may be adjusted in parallel to support the Treg cell expansionprocess in order to maintain lactate levels 7 mmol/L and to maintain thesingle cell suspension of the Treg culture by shearing cellmicro-colonies at an IC circulation rate of 300 mL/min through the lumenof the Quantum System HFM bioreactor under functional closed conditions.

Example 2

The tables below provide example task settings (e.g., flow rates,angular rotation, outlet, etc.) for different components (e.g., pumps,rocker, valves, etc.) of a cell expansion system over several days ofperforming an example protocol for the expansion of T cells. Theprotocol may follow the following sequence:

Day 0: Load Set, Prime, Add Media, Load Cells, and begin feeding

Day 3: Add a bolus of cytokines to the IC loop while re-distributing thecells. Begin feeding again.

Day 6: Add a bolus of cytokines to the IC loop while re-distributing thecells. Begin feeding again.

Day 9: Add a bolus of cytokines to the IC loop while re-distributing thecells. Begin feeding again.

Day 11-13: Harvest; reload remaining cells. Harvest (Day 14)

Table(s) of settings: changes made compared to example factory settingsare highlighted in bold and underline

TABLE 12 IL-2 Concentration and Amount in Complete Media, ExampleComplete Media Volume IL-2 IL-2 (mL) (IU/mL) (IU) 2000 200 4E+05

TABLE 13 Volumes of Bolus Additions and IL-2 Amounts, Example BolusAdditions Volume IL-2 (mL) (IU) Day 0 (cell load) 100 1E+05 Day 3 1502E+05 Day 6 150 2E+05 Day 9 150 4E+05

TABLE 14 Settings Day 0-Day 2, Example Day 0 Load cells with UniformCells Feed Load Cell Suspension in BR cells Expansion IC EC Conditionmedia (1e8 lymphocytes) Custom 1 Set Prime washout Step 1 Step 2 Step 1Step 2 Step 3 Step 1 Step 2 STEP 1 STEP 2 STEP 3 STEP 4 STEP 5 STEP 6STEP 7 STEP 8 STEP 9 STEP 10 Task IC inlet Default Default EC None NoneCell IC None IC IC Media Settings settings settings media Media Media ICinlet 100 0 0 25 25 0 100 0.1 rate IC circ −17 100 100 150 150 200 −70 1rate EC inlet EC EC EC None None None None None Media Media Media ECinlet 148 0.1 0.1 0 0 0 0 0 rate EC circ −1.7 250 30 30 30 30 30 100rate Outlet IC and EC outlet EC outlet EC EC EC EC EC outlet EC outletoutlet outlet outlet outlet Rocker In Stationary Stationary In In In InStationary Motion (0°) (0°) Motion Motion Motion Motion (0°) (−90°,(−90°, (−90°, (−90°, (−90°, 180°, 1 sec) 180°, 180°, 180°, 180°, 1 sec)1 sec) 1 sec) 1 sec) Stop Exchange Time (10 min) Manual Empty IC Time ICManual condition (2.5 IC (20-50 min) bag volume (2 min) Volume (4320min) volume; (22 ml) (120 mL) 2.5 EC volume) Extra Necessary NA 2 L 1300mL 6 mL/hr EC Media 100 mL 22 mL 120 mL 432 mL Information volume (PBS)EC IC IC Media Media Media Time 10 min 35 min 5 min 30-60 min 7 min 2min 3 days

TABLE 15 Settings Day 3-Day 5, Example Day 3 Cells in BR Feed cells AddBag Contents Custom 2 Step 1 Step 2 Step 1 Step 2 STEP 11 STEP 12 STEP13 STEP 14 Task IC inlet Reagent IC Media IC Media IC Media Settings ICinlet   30   30 100  0.1 rate IC circ 100 100 −70 −0.1 rate EC inletNone None None None EC inlet  0  0  0 0  rate EC circ 100 100  30 150   rate Outlet EC outlet EC outlet EC outlet EC outlet Rocker In MotionIn Motion In Motion In Motion ( −90°, 180°, ( −90°, 180°, (−90°, 180°,(0°, 180°, 1 sec ) 1 sec ) 1 sec) 3600 sec) Stop Empty IC Volume ICVolume Manual condition Bag (22 mL) (120 mL) (4320 min) Extra Necessary150 mL 22 mL IC 120 mL 432 mL IC Informati volume Media Media Time 5 min2 min 3 days

TABLE 16 Settings Day 6-Day 8, Example Day 6 Cells in Add Bag ContentsBR Feed cells Feed cells Feed cells Step 1 Custom 3 STEP Step 2 Step 1Step 2 Step 3 Step 4 15 STEP 16 STEP 17 STEP 18 STEP 20 STEP 22 TaskSettings IC inlet Reagent IC Media IC Media IC Media IC Media IC MediaIC inlet   30   30 100 0.1 0.1 0.1 rate IC circ 100 100 −70 −0.1 −0.1−0.1 rate EC inlet None None None EC Media EC Media EC Media EC inlet  0 0 0 0.1 0.2 0.3 rate EC circ 100 100 30 200 200 250 rate Outlet EC ECoutlet EC outlet EC outlet EC outlet EC outlet outlet Rocker InIn Motion In Motion In Motion In Motion In Motion Motion (−90°, (−90°,(0°, 180°, (0°, 180°, (0°, 180°, (−90°, 180°, 1 sec) 180°, 1 sec) 3600sec) 3600 sec) 3600 sec) 180°, 1 sec) Stop Empty IC IC Time Time Manualcondition Bag Volume Volume (1440 min) (1440 min) (22 mL) (120 mL) ExtraNecessary 150 mL 22 mL IC 120 mL 144 mL IC 144 mL IC 144 mL ICInformation volume Media Media Media Media 144 mL 288 mL 436 mL EC MediaEC Media EC Media Time 4 min 2 min 3 days

TABLE 17 Settings Day 9-Day 10, Example Day 9 Cells in BR Feed cellsFeed cells Add Bag Contents Custom 3 Step 1 Step 2 Step 1 Step 2 Step 3STEP 23 STEP 24 STEP 17 STEP 18 STEP 20 Task Settings IC inlet ReagentIC Media IC Media IC Media IC Media IC inlet   30   30 100 0.1 0.1 rateIC circ 100 100 −70 −0.1 −0.1 rate EC inlet None None None EC Media ECMedia EC inlet  0  0 0 0.1 0.2 rate EC circ 100 100 30 200 200 rateOutlet EC EC outlet EC outlet EC outlet EC outlet outlet Rocker InIn Motion In Motion In Motion In Motion Motion (−90°, (−90°, (0°, 180°,(0°, 180°, (−90°, 180°, 1 sec) 180°, 1 sec) 3600 sec) 3600 sec)180°, 1 sec) Stop Empty IC IC Time Time condition Bag Volume Volume(1440 min) (1440 min) (22 mL) (120 mL) Extra Necessary 150 mL 22 mL IC120 mL 144 mL IC 144 mL IC

volume Media Media Media 144 mL 288 mL EC Media EC Media Time 4 min 2min 2 days

indicates data missing or illegible when filed

TABLE 18 Settings Day 11, Example Harvest Cells in Mix Load cells withUniform Suspension BR Feed cells Custom 4 (Harvest Product) Custom 2Step 1 Harvest Step 1 Step 2 Step 3 Step 1 Step 2 STEP 31 STEP 32 STEP33 STEP 34 STEP 35 STEP 13 STEP 14 Task Settings IC inlet None EC CellIC Media None IC Media IC Media Media IC inlet 0 100 25 25 0 100 0.1rate IC circ rate 200 −20 150 150 200 −70 −0.1 EC inlet None EC NoneNone None None None Media EC inlet 0 100 0 0 0 0 0 rate EC circ rate 200  30 30 30 30 30 150 Outlet EC Harvest EC outlet EC outlet EC outlet ECoutlet EC outlet outlet Rocker In In In Motion In Motion In Motion InMotion In Motion Motion Motion (−90°, (−90°, 180°, (−90°, (−90°, (0°,180°, (−90°, (−90°, 180°, 1 sec) 1 sec) 180°, 1 sec) 180°, 1 sec) 3600sec) 180°, 1 sec) 180°, 1 sec) Stop Time (3 min) IC Empty IC volume Time(2 min) IC Manual condition Volume bag (22 ml) Volume (1440 min)(400 mL) (120 mL) Extra Necessary 800 mL (variable) 22 mL IC 120 mL 144mL IC Information volume EC mL Media Media Media Time 3 min 4 min 10 min2 min 1 days

TABLE 19 Settings Day 12, Example Harvest Cells in Mix Load cells withUniform Suspension BR Feed cells Custom 4 (Harvest Product) Custom 2Step 1 Harvest Step 1 Step 2 Step 3 Step 1 Step 2 STEP 31 STEP 32 STEP33 STEP 34 STEP 35 STEP 13 STEP 14 Task Settings IC inlet None EC CellIC Media None IC Media IC Media Media IC inlet 0 100 25 25 0 100 0.1rate IC circ rate 200 −20 150 150 200 −70 −0.1 EC inlet None EC NoneNone None None None Media EC inlet 0 100 0 0 0 0 0 rate EC circ rate 200  30 30 30 30 30 150 Outlet EC Harvest EC outlet EC outlet EC outlet ECoutlet EC outlet outlet Rocker In In In Motion In Motion In Motion InMotion In Motion Motion Motion (−90°, (−90°, 180°, (−90°, (−90°, (0°,180°, (−90°, (−90°, 180°, 1 sec) 1 sec) 180°, 1 sec) 180°, 1 sec) 3600sec) 180°, 1 sec) 180°, 1 sec) Stop Time (3 min) IC Empty IC volume Time(2 min) IC Manual condition Volume bag (22 ml) Volume (1440 min)(400 mL) (120 mL) Extra Necessary 800 mL (variable) 22 mL IC 120 mL 144mL IC Information volume EC mL Media Media Media Time 3 min 4 min 10 min2 min 1 days

TABLE 20 Settings Day 13, Example Harvest Cells in Mix Load cells withUniform Suspension BR Feed cells Custom 4 (Harvest Product) Custom 2Step 1 Harvest Step 1 Step 2 Step 3 Step 1 Step 2 STEP 31 STEP 32 STEP33 STEP 34 STEP 35 STEP 13 STEP 14 Task Settings IC inlet None EC CellIC Media None IC Media IC Media Media IC inlet 0 100 25 25 0 100 0.1rate In circ rate 200 −20 150 150 200 −70 −0.1 EC inlet None EC NoneNone None None None Media EC inlet 0 100 0 0 0 0 0 rate EC circ rate 200  30 30 30 30 30 150 Outlet EC Harvest EC EC outlet EC outlet EC outletEC outlet outlet outlet Rocker In In In In Motion In Motion In Motion InMotion Motion Motion Motion (−90°, 180°, (−90°, (−90°, (0°, 180°, (−90°,(−90°, (−90°, 1 sec) 180°, 1 sec) 180°, 1 sec) 3600 sec) 180°, 1 sec)180°, 1 sec) 180°, 1 sec) Stop Time (3 min) IC Empty IC volume Time (2min) IC Manual condition Volume bag (22 ml) Volume (1440 min) (400 mL)(120 mL) Extra Necessary 800 mL (variable) 22 mL IC 120 mL 144 mL ICInformation volume EC mL Media Media Media Time 3 min 4 min 10 min 2 min1 days

TABLE 21 Settings Day 14, Example Harvest Mix Custom 4 Step 1 HarvestSTEP 31 STEP 32 Task IC inlet None EC Media Settings IC inlet rate  0100 IC circ rate 200 −20 EC inlet None EC Media EC inlet rate  0 100 ECcirc rate 200   30 Outlet EC outlet Harvest Rocker In Motion In Motion(−90°, 180°, (−90°, 180°, 1 sec) 1 sec) Stop Time IC Volume condition (3min) ( 400 mL ) Extra Necessary 800 mL Information volume EC Media Time3 min 4 min

It will be apparent to those skilled in the art that variousmodifications and variations may be made to the methods and structure ofthe present invention without departing from its scope. Thus, it shouldbe understood that the invention is not to be limited to the specificexamples given. Rather, the invention is intended to cover modificationsand variations within the scope of the following claims and theirequivalents.

What is claimed is:
 1. A method of expanding cells in a cell expansionsystem, the method comprising: loading a first volume of fluidcomprising a plurality of cells into the cell expansion system, whereinthe cell expansion system comprises a cell growth chamber; loading asecond volume of fluid comprising media into a portion of a first fluidcirculation path to position the first volume of fluid in a firstportion of the cell growth chamber; feeding the cells; and expanding thecells.
 2. The method of claim 1, wherein the cell expansion systemcomprises a second fluid circulation path.
 3. The method of claim 1,wherein fluid in the first fluid circulation path flows through anintracapillary space of the cell growth chamber.
 4. The method of claim3, wherein fluid in the second fluid circulation path flows through anextracapillary space of the cell growth chamber.
 5. The method of claim1, wherein the first volume of fluid comprising the plurality of cellsis loaded without activating an intracapillary circulation pump.
 6. Themethod of claim 1, wherein the first volume of fluid is the same as thesecond volume of fluid.
 7. The method of claim 1, wherein the firstvolume of fluid is different from the second volume of fluid.
 8. Themethod of claim 1, wherein a sum of the first volume of fluid and thesecond volume of fluid is equal to a percentage of a volume of the firstfluid circulation path.
 9. The method of claim 8, wherein the sum of thefirst volume of fluid and the second volume of fluid is about fiftypercent of the volume of the first fluid circulation path.
 10. Themethod of claim 1, wherein the first portion of the cell growth chambercomprises about a central region of the cell growth chamber.
 11. Themethod of claim 1, wherein the plurality of cells comprise one or moresubpopulations of T cells.
 12. The method of claim 11, wherein the oneor more subpopulations of T cells comprise regulatory T cells (Tregs).13. The method of claim 1, wherein the cell growth chamber comprises ahollow fiber membrane.
 14. The method of claim 1, further comprising:stimulating the cells with an activator to activate the expanding of thecells.
 15. The method of claim 1, wherein a first portion of the firstfluid flow path between a connection and an inlet port of the cellgrowth chamber comprises a third volume.
 16. The method of claim 15,wherein the intracapillary space of the cell growth chamber comprises afourth volume.
 17. The method of claim 16, wherein the second volume offluid is at least as much as the third volume.
 18. The method of claim17, wherein the second volume of fluid is at least as much as a sum ofthe third volume and a percentage of the fourth volume that would not beoccupied by the first volume of fluid.
 19. The method of claim 18,wherein the percentage of the fourth volume comprises between about 5percent and about 50 percent.
 20. A method of expanding cells in a cellexpansion system, the method comprising: loading a fluid comprising aplurality of cells into a cell growth chamber in the cell expansionsystem; exposing the plurality of cells to an activator; expanding theplurality of cells during a first time period; and after expanding theplurality of cells during the first time period, circulating theplurality of cells at a first circulation rate during a second timeperiod to reduce a number of cells in a cell cluster.
 21. The method ofclaim 20, wherein the circulating the plurality of cells at the firstcirculation rate causes the cell cluster to incur a shear stress. 22.The method of claim 21, wherein the shear stress causes at least a firstcell in the cell cluster to break apart from the cell cluster.
 23. Themethod of claim 20, wherein the reducing the number of cells in the cellcluster reduces a size of the cell cluster to a reduced size.
 24. Themethod of claim 23, wherein the reduced size of the cell cluster isbetween about 25 microns and about 300 microns.
 25. The method of claim24, wherein the reduced size of the cell cluster is between about 50microns and about 250 microns.
 26. The method of claim 25, wherein thereduced size of the cell cluster is between about 75 microns and about200 microns.
 27. The method of claim 23, wherein the reduced size of thecell cluster is less than about 200 microns.
 28. The method of claim 20,wherein the plurality of cells comprise regulatory T cells (Tregs). 29.The method of claim 20, after the second time period, moving cells notpositioned in the cell growth chamber back into the cell growth chamberduring a third time period.
 30. The method of claim 29, after the thirdtime period, expanding the plurality of cells during a fourth timeperiod; and after expanding the plurality of cells during the fourthtime period, circulating the plurality of cells at the first circulationrate during a fifth time period to reduce a second number of cells in asecond cell cluster; after the fifth time period, moving second cellsnot positioned in the cell growth chamber back into the cell growthchamber during a sixth time period.